genomic dna  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97

    Structured Review

    Thermo Fisher genomic dna
    Tumorigenesis after “active” transfection with <t>DNA</t> supernatant of <t>SW480</t> cells and passive transfection using DNA-depleted supernatant. A. Agarose gel electrophoresis of DNA extracted from supernatant (Sp), DNA from supernatant treated with DNAse I (Sp+D), protease only (Sp+P), and both (Sp+D+P). DNA is partially degraded by DNAse I and protease, but fully degraded when exposed to both treatments. B. No tumor growth was observed in NIH3T3 exposed to DNA-depleted (DNAse I/Prot) supernatant of SW480 cells, while passively transfected NIH3T3 with untreated supernatant are tumorigenic. +Ctr are SW480 cells and −Ctr are NIH3T3 cells.
    Genomic Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genomic dna/product/Thermo Fisher
    Average 97 stars, based on 1244 article reviews
    Price from $9.99 to $1999.99
    genomic dna - by Bioz Stars, 2020-01
    97/100 stars

    Images

    1) Product Images from "Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model"

    Article Title: Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052754

    Tumorigenesis after “active” transfection with DNA supernatant of SW480 cells and passive transfection using DNA-depleted supernatant. A. Agarose gel electrophoresis of DNA extracted from supernatant (Sp), DNA from supernatant treated with DNAse I (Sp+D), protease only (Sp+P), and both (Sp+D+P). DNA is partially degraded by DNAse I and protease, but fully degraded when exposed to both treatments. B. No tumor growth was observed in NIH3T3 exposed to DNA-depleted (DNAse I/Prot) supernatant of SW480 cells, while passively transfected NIH3T3 with untreated supernatant are tumorigenic. +Ctr are SW480 cells and −Ctr are NIH3T3 cells.
    Figure Legend Snippet: Tumorigenesis after “active” transfection with DNA supernatant of SW480 cells and passive transfection using DNA-depleted supernatant. A. Agarose gel electrophoresis of DNA extracted from supernatant (Sp), DNA from supernatant treated with DNAse I (Sp+D), protease only (Sp+P), and both (Sp+D+P). DNA is partially degraded by DNAse I and protease, but fully degraded when exposed to both treatments. B. No tumor growth was observed in NIH3T3 exposed to DNA-depleted (DNAse I/Prot) supernatant of SW480 cells, while passively transfected NIH3T3 with untreated supernatant are tumorigenic. +Ctr are SW480 cells and −Ctr are NIH3T3 cells.

    Techniques Used: Transfection, Agarose Gel Electrophoresis

    Human DNA transfer in rat colon tumors by PCR-sequencing. Representative pictures of PCR detection of a repetitive sequence of rat ( LINE 1 ) in a rat tumor (DMH and DMH+SW480). ( A ) Alu Yd6 human sequences were only amplified from the colon tumors of DMH+SW480-treated rats. Rat tail and human cells were used as positive and negative controls. Human K-ras and RAB30 genes were only detected in the tumors of rats receiving DMH and SW480 cells. ( B ) Sequence analysis of the PCR product of RAB30 in a colon tumor treated with DMH+SW480 cells. Arrows indicate the position where the nucleotide sequence is different between species and clearly shows the existence of both sequences. Human SW480 cells (control).
    Figure Legend Snippet: Human DNA transfer in rat colon tumors by PCR-sequencing. Representative pictures of PCR detection of a repetitive sequence of rat ( LINE 1 ) in a rat tumor (DMH and DMH+SW480). ( A ) Alu Yd6 human sequences were only amplified from the colon tumors of DMH+SW480-treated rats. Rat tail and human cells were used as positive and negative controls. Human K-ras and RAB30 genes were only detected in the tumors of rats receiving DMH and SW480 cells. ( B ) Sequence analysis of the PCR product of RAB30 in a colon tumor treated with DMH+SW480 cells. Arrows indicate the position where the nucleotide sequence is different between species and clearly shows the existence of both sequences. Human SW480 cells (control).

    Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification

    Tumorigenesis and DNA transfer in recipient murine NIH3T3 cells after passive transfection. A. Tumor growth in nude mice from “passively” transformed cells. Faster and higher tumor growth was observed in SB1 pool and CCPS pool (NIH3T3 exposed to supernatant of SW480 cells and to the serum of a patient with colon cancer, respectively). SW480 cells were used as positive control, whereas NIH3T3 and NIH3T3 exposed to normal serum showed essentially no growth . B. Representative pictures of tumors in mice from each group. C. Southern blot hybridization of SB1 and CCPS pools of cells against genomic DNA of SW480 cells. Lane SW480 cells are the positive control and NIH3T3, the negative one. A clear hybridization signal is only observed in SB1 and CCPS lanes. D. FISH analysis of repetitive human sequences. Positive control is human lymphocytes and murine cells negative control. SB1 cells shows strong signal. E. Tumor growth is similar in NIH3T3 actively transfected with genomic DNA from SW480 cells (Neo-Geno) and actively transfected DNA extracted from supernatant of SW480 cells as compared with no growth in NIH3T3 (-Crt) and transfected with the empty-vector only. Positive control, SW480 cells.
    Figure Legend Snippet: Tumorigenesis and DNA transfer in recipient murine NIH3T3 cells after passive transfection. A. Tumor growth in nude mice from “passively” transformed cells. Faster and higher tumor growth was observed in SB1 pool and CCPS pool (NIH3T3 exposed to supernatant of SW480 cells and to the serum of a patient with colon cancer, respectively). SW480 cells were used as positive control, whereas NIH3T3 and NIH3T3 exposed to normal serum showed essentially no growth . B. Representative pictures of tumors in mice from each group. C. Southern blot hybridization of SB1 and CCPS pools of cells against genomic DNA of SW480 cells. Lane SW480 cells are the positive control and NIH3T3, the negative one. A clear hybridization signal is only observed in SB1 and CCPS lanes. D. FISH analysis of repetitive human sequences. Positive control is human lymphocytes and murine cells negative control. SB1 cells shows strong signal. E. Tumor growth is similar in NIH3T3 actively transfected with genomic DNA from SW480 cells (Neo-Geno) and actively transfected DNA extracted from supernatant of SW480 cells as compared with no growth in NIH3T3 (-Crt) and transfected with the empty-vector only. Positive control, SW480 cells.

    Techniques Used: Transfection, Mouse Assay, Transformation Assay, Positive Control, Southern Blot, Hybridization, Fluorescence In Situ Hybridization, Negative Control, Plasmid Preparation

    Human DNA transfer in rat colon tumors by FISH. Representative photographs FISH analyses of rat repetitive sequences ( LINE 1 ) and human ( Alu Yd6 ) in a colon tumor from a rat treated with DMH+SW480 cells ( A ) . Pictures at left (blue) are nuclei stained with DAPI, green are rat specific LINE 1 signals. Red are human Alu Yd6 signals and orange are cells showing both signals. B . Representative photographs of a colon tumor from a rat treated with DMH only. Absent red signals represent the lack of human sequences in these cells. Co-incubation with both probes at the right confirms the lack of DNA transfer.
    Figure Legend Snippet: Human DNA transfer in rat colon tumors by FISH. Representative photographs FISH analyses of rat repetitive sequences ( LINE 1 ) and human ( Alu Yd6 ) in a colon tumor from a rat treated with DMH+SW480 cells ( A ) . Pictures at left (blue) are nuclei stained with DAPI, green are rat specific LINE 1 signals. Red are human Alu Yd6 signals and orange are cells showing both signals. B . Representative photographs of a colon tumor from a rat treated with DMH only. Absent red signals represent the lack of human sequences in these cells. Co-incubation with both probes at the right confirms the lack of DNA transfer.

    Techniques Used: Fluorescence In Situ Hybridization, Staining, Incubation

    DNA copy number analysis of extracellular and intracellular DNA from SW480 cells and gene transfer to murine NIH3T3 cells. A. Heat map representing the DNA copy number along chromosome 8. Blue represents regions with deletions and red regions with amplifications. A nearly identical pattern of DNA copy number changes between extracellular (SpDNA SW480) and intracellular (DNA SW480) DNA, compared to a common normal reference can be observed. RT-PCR, PCR and sequencing of Human K-ras ( B ) and RAB30 ( C ) . Negative control was NIH3T3 cells.
    Figure Legend Snippet: DNA copy number analysis of extracellular and intracellular DNA from SW480 cells and gene transfer to murine NIH3T3 cells. A. Heat map representing the DNA copy number along chromosome 8. Blue represents regions with deletions and red regions with amplifications. A nearly identical pattern of DNA copy number changes between extracellular (SpDNA SW480) and intracellular (DNA SW480) DNA, compared to a common normal reference can be observed. RT-PCR, PCR and sequencing of Human K-ras ( B ) and RAB30 ( C ) . Negative control was NIH3T3 cells.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Negative Control

    2) Product Images from "Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model"

    Article Title: Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052754

    Tumorigenesis after “active” transfection with DNA supernatant of SW480 cells and passive transfection using DNA-depleted supernatant. A. Agarose gel electrophoresis of DNA extracted from supernatant (Sp), DNA from supernatant treated with DNAse I (Sp+D), protease only (Sp+P), and both (Sp+D+P). DNA is partially degraded by DNAse I and protease, but fully degraded when exposed to both treatments. B. No tumor growth was observed in NIH3T3 exposed to DNA-depleted (DNAse I/Prot) supernatant of SW480 cells, while passively transfected NIH3T3 with untreated supernatant are tumorigenic. +Ctr are SW480 cells and −Ctr are NIH3T3 cells.
    Figure Legend Snippet: Tumorigenesis after “active” transfection with DNA supernatant of SW480 cells and passive transfection using DNA-depleted supernatant. A. Agarose gel electrophoresis of DNA extracted from supernatant (Sp), DNA from supernatant treated with DNAse I (Sp+D), protease only (Sp+P), and both (Sp+D+P). DNA is partially degraded by DNAse I and protease, but fully degraded when exposed to both treatments. B. No tumor growth was observed in NIH3T3 exposed to DNA-depleted (DNAse I/Prot) supernatant of SW480 cells, while passively transfected NIH3T3 with untreated supernatant are tumorigenic. +Ctr are SW480 cells and −Ctr are NIH3T3 cells.

    Techniques Used: Transfection, Agarose Gel Electrophoresis

    Human DNA transfer in rat colon tumors by PCR-sequencing. Representative pictures of PCR detection of a repetitive sequence of rat ( LINE 1 ) in a rat tumor (DMH and DMH+SW480). ( A ) Alu Yd6 human sequences were only amplified from the colon tumors of DMH+SW480-treated rats. Rat tail and human cells were used as positive and negative controls. Human K-ras and RAB30 genes were only detected in the tumors of rats receiving DMH and SW480 cells. ( B ) Sequence analysis of the PCR product of RAB30 in a colon tumor treated with DMH+SW480 cells. Arrows indicate the position where the nucleotide sequence is different between species and clearly shows the existence of both sequences. Human SW480 cells (control).
    Figure Legend Snippet: Human DNA transfer in rat colon tumors by PCR-sequencing. Representative pictures of PCR detection of a repetitive sequence of rat ( LINE 1 ) in a rat tumor (DMH and DMH+SW480). ( A ) Alu Yd6 human sequences were only amplified from the colon tumors of DMH+SW480-treated rats. Rat tail and human cells were used as positive and negative controls. Human K-ras and RAB30 genes were only detected in the tumors of rats receiving DMH and SW480 cells. ( B ) Sequence analysis of the PCR product of RAB30 in a colon tumor treated with DMH+SW480 cells. Arrows indicate the position where the nucleotide sequence is different between species and clearly shows the existence of both sequences. Human SW480 cells (control).

    Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification

    Tumorigenesis and DNA transfer in recipient murine NIH3T3 cells after passive transfection. A. Tumor growth in nude mice from “passively” transformed cells. Faster and higher tumor growth was observed in SB1 pool and CCPS pool (NIH3T3 exposed to supernatant of SW480 cells and to the serum of a patient with colon cancer, respectively). SW480 cells were used as positive control, whereas NIH3T3 and NIH3T3 exposed to normal serum showed essentially no growth . B. Representative pictures of tumors in mice from each group. C. Southern blot hybridization of SB1 and CCPS pools of cells against genomic DNA of SW480 cells. Lane SW480 cells are the positive control and NIH3T3, the negative one. A clear hybridization signal is only observed in SB1 and CCPS lanes. D. FISH analysis of repetitive human sequences. Positive control is human lymphocytes and murine cells negative control. SB1 cells shows strong signal. E. Tumor growth is similar in NIH3T3 actively transfected with genomic DNA from SW480 cells (Neo-Geno) and actively transfected DNA extracted from supernatant of SW480 cells as compared with no growth in NIH3T3 (-Crt) and transfected with the empty-vector only. Positive control, SW480 cells.
    Figure Legend Snippet: Tumorigenesis and DNA transfer in recipient murine NIH3T3 cells after passive transfection. A. Tumor growth in nude mice from “passively” transformed cells. Faster and higher tumor growth was observed in SB1 pool and CCPS pool (NIH3T3 exposed to supernatant of SW480 cells and to the serum of a patient with colon cancer, respectively). SW480 cells were used as positive control, whereas NIH3T3 and NIH3T3 exposed to normal serum showed essentially no growth . B. Representative pictures of tumors in mice from each group. C. Southern blot hybridization of SB1 and CCPS pools of cells against genomic DNA of SW480 cells. Lane SW480 cells are the positive control and NIH3T3, the negative one. A clear hybridization signal is only observed in SB1 and CCPS lanes. D. FISH analysis of repetitive human sequences. Positive control is human lymphocytes and murine cells negative control. SB1 cells shows strong signal. E. Tumor growth is similar in NIH3T3 actively transfected with genomic DNA from SW480 cells (Neo-Geno) and actively transfected DNA extracted from supernatant of SW480 cells as compared with no growth in NIH3T3 (-Crt) and transfected with the empty-vector only. Positive control, SW480 cells.

    Techniques Used: Transfection, Mouse Assay, Transformation Assay, Positive Control, Southern Blot, Hybridization, Fluorescence In Situ Hybridization, Negative Control, Plasmid Preparation

    Human DNA transfer in rat colon tumors by FISH. Representative photographs FISH analyses of rat repetitive sequences ( LINE 1 ) and human ( Alu Yd6 ) in a colon tumor from a rat treated with DMH+SW480 cells ( A ) . Pictures at left (blue) are nuclei stained with DAPI, green are rat specific LINE 1 signals. Red are human Alu Yd6 signals and orange are cells showing both signals. B . Representative photographs of a colon tumor from a rat treated with DMH only. Absent red signals represent the lack of human sequences in these cells. Co-incubation with both probes at the right confirms the lack of DNA transfer.
    Figure Legend Snippet: Human DNA transfer in rat colon tumors by FISH. Representative photographs FISH analyses of rat repetitive sequences ( LINE 1 ) and human ( Alu Yd6 ) in a colon tumor from a rat treated with DMH+SW480 cells ( A ) . Pictures at left (blue) are nuclei stained with DAPI, green are rat specific LINE 1 signals. Red are human Alu Yd6 signals and orange are cells showing both signals. B . Representative photographs of a colon tumor from a rat treated with DMH only. Absent red signals represent the lack of human sequences in these cells. Co-incubation with both probes at the right confirms the lack of DNA transfer.

    Techniques Used: Fluorescence In Situ Hybridization, Staining, Incubation

    DNA copy number analysis of extracellular and intracellular DNA from SW480 cells and gene transfer to murine NIH3T3 cells. A. Heat map representing the DNA copy number along chromosome 8. Blue represents regions with deletions and red regions with amplifications. A nearly identical pattern of DNA copy number changes between extracellular (SpDNA SW480) and intracellular (DNA SW480) DNA, compared to a common normal reference can be observed. RT-PCR, PCR and sequencing of Human K-ras ( B ) and RAB30 ( C ) . Negative control was NIH3T3 cells.
    Figure Legend Snippet: DNA copy number analysis of extracellular and intracellular DNA from SW480 cells and gene transfer to murine NIH3T3 cells. A. Heat map representing the DNA copy number along chromosome 8. Blue represents regions with deletions and red regions with amplifications. A nearly identical pattern of DNA copy number changes between extracellular (SpDNA SW480) and intracellular (DNA SW480) DNA, compared to a common normal reference can be observed. RT-PCR, PCR and sequencing of Human K-ras ( B ) and RAB30 ( C ) . Negative control was NIH3T3 cells.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Negative Control

    3) Product Images from "Single molecule analysis of Trypanosoma brucei DNA replication dynamics"

    Article Title: Single molecule analysis of Trypanosoma brucei DNA replication dynamics

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1389

    The T. brucei replication rate is similar to rates of other eukaryotes. ( A ) Schematic representation of replication analysis by sequential pulse labeling. Actively replicating DNA in exponentially growing cells is labeled by performing consecutive pulses of growth in the presence of thymidine analogs IdU or CldU. The incorporated analogs are identified by immunostaining, appearing as red (IdU) or green (CldU) signals. The temporal order of replication is established by the sequence of labeling (red followed by green). The resulting distinct labeling patterns in individual molecules identify replication forks active during the transition from IdU (red) to CldU pulsing (green) (a), as well as replication initiation (b) and termination events (c). ( B ) IdU and CldU detection on T. brucei procyclic form. IdU in red, CldU in green and DAPI in blue. N is the nucleus and k is the kinetoplast (the DNA from the single mitochondria). ( C and D ) Nucleoside-labeled genomic DNA (gDNA) from two different forms of T. brucei were digested with the restriction enzyme Asc I and separated by PFGE using a CHEF Mapper (BioRad), under the following conditions: two-state program, 120° included angle, 6 V/cm, 18 s initial pulse, 33 s final pulse, 30 h running time, linear ramping. L-Lambda Ladder PFG marker (NEB); 1-digested DNA from one agarose plug and 2-digested DNA from four agarose plugs, where a region was cut out for DNA isolation. The yellow boxes indicate the region from where the DNA was recovered. ( E and F ) The DNA molecules were stretched on silanized glass slides and nucleoside labeling detected with antibodies against IdU (red), CldU (green) and single-stranded DNA (white). ( E ) Alignment of DNA molecules from procyclic form of 145 kb average length size. ( F ) Alignment of DNA molecules from bloodstream form of 157 kb average size length. These molecules were used to estimate the average replication rate.
    Figure Legend Snippet: The T. brucei replication rate is similar to rates of other eukaryotes. ( A ) Schematic representation of replication analysis by sequential pulse labeling. Actively replicating DNA in exponentially growing cells is labeled by performing consecutive pulses of growth in the presence of thymidine analogs IdU or CldU. The incorporated analogs are identified by immunostaining, appearing as red (IdU) or green (CldU) signals. The temporal order of replication is established by the sequence of labeling (red followed by green). The resulting distinct labeling patterns in individual molecules identify replication forks active during the transition from IdU (red) to CldU pulsing (green) (a), as well as replication initiation (b) and termination events (c). ( B ) IdU and CldU detection on T. brucei procyclic form. IdU in red, CldU in green and DAPI in blue. N is the nucleus and k is the kinetoplast (the DNA from the single mitochondria). ( C and D ) Nucleoside-labeled genomic DNA (gDNA) from two different forms of T. brucei were digested with the restriction enzyme Asc I and separated by PFGE using a CHEF Mapper (BioRad), under the following conditions: two-state program, 120° included angle, 6 V/cm, 18 s initial pulse, 33 s final pulse, 30 h running time, linear ramping. L-Lambda Ladder PFG marker (NEB); 1-digested DNA from one agarose plug and 2-digested DNA from four agarose plugs, where a region was cut out for DNA isolation. The yellow boxes indicate the region from where the DNA was recovered. ( E and F ) The DNA molecules were stretched on silanized glass slides and nucleoside labeling detected with antibodies against IdU (red), CldU (green) and single-stranded DNA (white). ( E ) Alignment of DNA molecules from procyclic form of 145 kb average length size. ( F ) Alignment of DNA molecules from bloodstream form of 157 kb average size length. These molecules were used to estimate the average replication rate.

    Techniques Used: Labeling, Immunostaining, Sequencing, Marker, DNA Extraction

    Chromosome 1 replication in unperturbed cells. T. brucei replication was mapped by MFA using quantitative PCR, and compared to MFAseq. The line graph shows qPCR at a number of loci within a central 347 kb region of chromosome 1: at each locus the relative quantity of early S phase (blue), late S phase (orange) and G2 phase (black) DNA is shown, in all cases normalized to qPCR of a region of kinetoplast DNA (not shown); G2 values at each locus are set at 1, and the S phase samples shown as a proportion of that value (error bars indicate standard deviation from three experimental repeats). Locations of the qPCR loci within the chromosome (bp) are shown (points in the graph from 5′ to 3′ indicate amplified regions from chromosome 1: nt 397 255–397 535, nt 440 426–440 525, nt 463 829–463 933, nt 504 971–505 071, nt 549 145–549 244, nt 664 782–664 876 and nt 797 547–797 644). MFAseq mapping is shown as dots across the whole chromosome: the ratio of the read-depth between the early (light blue) or late S phase (light orange) and G2 samples is shown, where each dot represents 2500 bp.
    Figure Legend Snippet: Chromosome 1 replication in unperturbed cells. T. brucei replication was mapped by MFA using quantitative PCR, and compared to MFAseq. The line graph shows qPCR at a number of loci within a central 347 kb region of chromosome 1: at each locus the relative quantity of early S phase (blue), late S phase (orange) and G2 phase (black) DNA is shown, in all cases normalized to qPCR of a region of kinetoplast DNA (not shown); G2 values at each locus are set at 1, and the S phase samples shown as a proportion of that value (error bars indicate standard deviation from three experimental repeats). Locations of the qPCR loci within the chromosome (bp) are shown (points in the graph from 5′ to 3′ indicate amplified regions from chromosome 1: nt 397 255–397 535, nt 440 426–440 525, nt 463 829–463 933, nt 504 971–505 071, nt 549 145–549 244, nt 664 782–664 876 and nt 797 547–797 644). MFAseq mapping is shown as dots across the whole chromosome: the ratio of the read-depth between the early (light blue) or late S phase (light orange) and G2 samples is shown, where each dot represents 2500 bp.

    Techniques Used: Real-time Polymerase Chain Reaction, Standard Deviation, Amplification

    Chromosome 1 replication under replicative stress. T. brucei procyclic cells were treated with HU or maintained in culture as control (−). After 12 h of treatment, cells were washed (0 h) and maintained in culture for 3 h. ( A ) Cells were then stained with propidium iodide and analyzed by FACS. ( B ) Additionally, DNA from synchronized cells was extracted and quantified by real time PCR (normalized by regions from kinetoplast DNA) using primers to amplify the regions of chromosome 1 (points in the graph from 5′ to 3′ indicate amplified regions from chromosome 1: nt 397 255–397 535, nt 440 426–440 525, nt 463 829–463 933, nt 504 971–505 071, nt 549 145–549 244, nt 664 782–664 876 and nt 797 547–797 644). P -values from Student's t -test are * P
    Figure Legend Snippet: Chromosome 1 replication under replicative stress. T. brucei procyclic cells were treated with HU or maintained in culture as control (−). After 12 h of treatment, cells were washed (0 h) and maintained in culture for 3 h. ( A ) Cells were then stained with propidium iodide and analyzed by FACS. ( B ) Additionally, DNA from synchronized cells was extracted and quantified by real time PCR (normalized by regions from kinetoplast DNA) using primers to amplify the regions of chromosome 1 (points in the graph from 5′ to 3′ indicate amplified regions from chromosome 1: nt 397 255–397 535, nt 440 426–440 525, nt 463 829–463 933, nt 504 971–505 071, nt 549 145–549 244, nt 664 782–664 876 and nt 797 547–797 644). P -values from Student's t -test are * P

    Techniques Used: Staining, FACS, Real-time Polymerase Chain Reaction, Amplification

    DNA replication rate in procyclic and bloodstream life cycle forms
    Figure Legend Snippet: DNA replication rate in procyclic and bloodstream life cycle forms

    Techniques Used:

    Identification and isolation of a 347 kb fragment from chromosome 1 of T. brucei procyclic form. A . Schematic representation of SMARD methodology. Consecutive 40 min pulses of thymidine analogs IdU and CldU are performed on an exponential growing cell culture. The cells were cast into agarose plugs then treated with proteinase K. The DNA remaining in the agarose plug was digested with the restriction enzyme Asc I and subjected to PFGE. Part of the pulse field gel was transferred to nylon membrane, which was hybridized with a probe specific for the target fragment. A gel slice containing the DNA of interest was excised from the gel and after the agarose was melted and digested the DNA was stretched on silanized slides. The slides were hybridized with specific FISH probes to identify the target fragment. IdU, CldU and FISH probes were detected by indirect immunofluorescence using specific antibodies. Red, green and blue regions on the DNA molecule are IdU, CldU and FISH probe signals, respectively. ( B ) Schematic map of T. brucei chromosome 1 (∼1.06 Mb in length) and the 347 kb fragment obtained by Asc I digestion of chromosome 1. The centromere (from ∼760 to 790 kb) is marked by a green box. The blue boxes under the 347 kb fragment represent the probes used to identify this fragment on slides prepared for SMARD. The asterisk shows the region targeted by the specific probe used to identify the 347 kb fragment on Southern blots. ( C ) The same pulsed field gel from Figure 1C was transferred to a nylon membrane and ( D ) hybridized with a specific probe to identify the 347 kb fragment. The gel was cut into slices (a, b and c) in order to enrich for the 347 kb fragment. ( E ) PCR was performed on the DNA from the slices using a specific pair of primers to identify the 347 kb fragment. ( C and E ) The yellow arrow indicates the position where the 347 kb fragment was detected.
    Figure Legend Snippet: Identification and isolation of a 347 kb fragment from chromosome 1 of T. brucei procyclic form. A . Schematic representation of SMARD methodology. Consecutive 40 min pulses of thymidine analogs IdU and CldU are performed on an exponential growing cell culture. The cells were cast into agarose plugs then treated with proteinase K. The DNA remaining in the agarose plug was digested with the restriction enzyme Asc I and subjected to PFGE. Part of the pulse field gel was transferred to nylon membrane, which was hybridized with a probe specific for the target fragment. A gel slice containing the DNA of interest was excised from the gel and after the agarose was melted and digested the DNA was stretched on silanized slides. The slides were hybridized with specific FISH probes to identify the target fragment. IdU, CldU and FISH probes were detected by indirect immunofluorescence using specific antibodies. Red, green and blue regions on the DNA molecule are IdU, CldU and FISH probe signals, respectively. ( B ) Schematic map of T. brucei chromosome 1 (∼1.06 Mb in length) and the 347 kb fragment obtained by Asc I digestion of chromosome 1. The centromere (from ∼760 to 790 kb) is marked by a green box. The blue boxes under the 347 kb fragment represent the probes used to identify this fragment on slides prepared for SMARD. The asterisk shows the region targeted by the specific probe used to identify the 347 kb fragment on Southern blots. ( C ) The same pulsed field gel from Figure 1C was transferred to a nylon membrane and ( D ) hybridized with a specific probe to identify the 347 kb fragment. The gel was cut into slices (a, b and c) in order to enrich for the 347 kb fragment. ( E ) PCR was performed on the DNA from the slices using a specific pair of primers to identify the 347 kb fragment. ( C and E ) The yellow arrow indicates the position where the 347 kb fragment was detected.

    Techniques Used: Isolation, Cell Culture, Fluorescence In Situ Hybridization, Immunofluorescence, Pulsed-Field Gel, Polymerase Chain Reaction

    4) Product Images from "The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions"

    Article Title: The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006875

    TcRad51 immunolocalization in T . cruzi . A) WT, TcRAD51 +/- and TcRAD51 ox cells are shown before irradiation (non-irradiated) and 24 h after exposure to 500 Gy of gamma irradiation. DNA is shown stained with DAPI and TcRad51 was detected using anti-TcRad51 antibody raised in mouse (diluted 1:2,000) and visualized with Alexa 555 conjugated goat-derived anti-mouse IgG secondary (diluted 1:5,000); black bar: 5 μm. Cells are also shown in phase contrast but at a lower magnification (white bar: 2 μm). A graphical representation of fluorescence intensities is shown alongside the images. Vertical bars indicate standard deviation, asterisks represent statistically significant differences (*** p
    Figure Legend Snippet: TcRad51 immunolocalization in T . cruzi . A) WT, TcRAD51 +/- and TcRAD51 ox cells are shown before irradiation (non-irradiated) and 24 h after exposure to 500 Gy of gamma irradiation. DNA is shown stained with DAPI and TcRad51 was detected using anti-TcRad51 antibody raised in mouse (diluted 1:2,000) and visualized with Alexa 555 conjugated goat-derived anti-mouse IgG secondary (diluted 1:5,000); black bar: 5 μm. Cells are also shown in phase contrast but at a lower magnification (white bar: 2 μm). A graphical representation of fluorescence intensities is shown alongside the images. Vertical bars indicate standard deviation, asterisks represent statistically significant differences (*** p

    Techniques Used: Irradiation, Staining, Derivative Assay, Fluorescence, Standard Deviation

    T . cruzi response to treatment with DNA cross-linking agents. Sensitivity of WT, TcRAD51 ox and TcRAD51 -/+ cells to: (A) 0, 500 J/m 2 or 1.000 J/m 2 of UV-light, or (B) 25 μM, 50 μM or 75 μM cisplatin. Parasites were counted 48 h after treatment. Numbers are represented as a percentage of untreated cells. Values represent the mean of triplicates. Error bars indicate standard deviations.
    Figure Legend Snippet: T . cruzi response to treatment with DNA cross-linking agents. Sensitivity of WT, TcRAD51 ox and TcRAD51 -/+ cells to: (A) 0, 500 J/m 2 or 1.000 J/m 2 of UV-light, or (B) 25 μM, 50 μM or 75 μM cisplatin. Parasites were counted 48 h after treatment. Numbers are represented as a percentage of untreated cells. Values represent the mean of triplicates. Error bars indicate standard deviations.

    Techniques Used:

    Growth of T . cruzi following DNA damage treatment. A) The sensitivity of WT and TcRAD51 -/+ cells to gamma radiation was determined. Parasites were exposed to 0 Gy or 500 Gy of gamma irradiation, the number of parasites was determined every two days for 28 d. Values represent the mean of triplicates and error bars indicate standard deviations. B) Chromosomal profile from different T . cruzi (WT, TcRAD51 ox, and TcRAD51 +/- ) samples after treatment with gamma radiation (500 Gy). Chromosomal bands from different T . cruzi samples were separated by PFGE and stained using ethidium bromide. Whole chromosomes from Saccharomyces cerevisiae were used as molecular weight markers. (C) T . cruzi growth curve after treatment with 20 mM HU. Arrow indicates the point when the drug was removed. (D) T . cruzi growth curve after treatment with 1.5 mM MMS. The number of cells was determined every 24 h by vital staining. Curves shown are an example of three independent experiments that were performed in triplicate. Error bars represent standard deviations. (E) Detection of H2A levels in epimastigotes treated with 20 mM HU. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot using anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%). (F) Detection of H2A levels in epimastigotes treated with 1.5 mM MMS. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot with anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%).
    Figure Legend Snippet: Growth of T . cruzi following DNA damage treatment. A) The sensitivity of WT and TcRAD51 -/+ cells to gamma radiation was determined. Parasites were exposed to 0 Gy or 500 Gy of gamma irradiation, the number of parasites was determined every two days for 28 d. Values represent the mean of triplicates and error bars indicate standard deviations. B) Chromosomal profile from different T . cruzi (WT, TcRAD51 ox, and TcRAD51 +/- ) samples after treatment with gamma radiation (500 Gy). Chromosomal bands from different T . cruzi samples were separated by PFGE and stained using ethidium bromide. Whole chromosomes from Saccharomyces cerevisiae were used as molecular weight markers. (C) T . cruzi growth curve after treatment with 20 mM HU. Arrow indicates the point when the drug was removed. (D) T . cruzi growth curve after treatment with 1.5 mM MMS. The number of cells was determined every 24 h by vital staining. Curves shown are an example of three independent experiments that were performed in triplicate. Error bars represent standard deviations. (E) Detection of H2A levels in epimastigotes treated with 20 mM HU. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot using anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%). (F) Detection of H2A levels in epimastigotes treated with 1.5 mM MMS. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot with anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%).

    Techniques Used: Irradiation, Staining, Molecular Weight, SDS Page, Western Blot

    Generation of TcRAD51 -/+ knockouts mutants and TcRad51 protein levels. (A) Ethidium bromide stained gel showing PCR products generated with primers represented indicated in the schematic representation. PCR was performed using genomic DNA of WT and TcRAD51 +/- epimastigotes. Upper panel: schematic representation of RAD51 and hygromycin allele detection. Note that the amplicon is only synthesized if the pair of primers simultaneously hybridizes inside and outside the deletion cassette. The figure is not on scale. (B) Schematic representation of the Southern blot analysis showing BglI sites and the probes used (C) Left panel: Purified HYGB and RAD51 fragments generated by PCR-amplification. Right panel: Southern blot analysis of BglI- and BglI-digested genomic DNA from WT and TcRAD51 +/- cells and probed with HYGB and RAD51 fragments. Ethidium bromide-stained agarose gel showing the digestion products that were further analyzed by Southern blotting. (D) Detection of TcRad51 levels in epimastigotes protein extracts from WT, TcRAD51 ox, and TcRAD51 -/+ cells. Cellular lysates were separated by SDS-PAGE, and proteins were detected by Western blot with anti-TcRad51 (1:2,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). A control showing tubulin levels was performed using mouse anti-tubulin (1:12,000) antiserum.
    Figure Legend Snippet: Generation of TcRAD51 -/+ knockouts mutants and TcRad51 protein levels. (A) Ethidium bromide stained gel showing PCR products generated with primers represented indicated in the schematic representation. PCR was performed using genomic DNA of WT and TcRAD51 +/- epimastigotes. Upper panel: schematic representation of RAD51 and hygromycin allele detection. Note that the amplicon is only synthesized if the pair of primers simultaneously hybridizes inside and outside the deletion cassette. The figure is not on scale. (B) Schematic representation of the Southern blot analysis showing BglI sites and the probes used (C) Left panel: Purified HYGB and RAD51 fragments generated by PCR-amplification. Right panel: Southern blot analysis of BglI- and BglI-digested genomic DNA from WT and TcRAD51 +/- cells and probed with HYGB and RAD51 fragments. Ethidium bromide-stained agarose gel showing the digestion products that were further analyzed by Southern blotting. (D) Detection of TcRad51 levels in epimastigotes protein extracts from WT, TcRAD51 ox, and TcRAD51 -/+ cells. Cellular lysates were separated by SDS-PAGE, and proteins were detected by Western blot with anti-TcRad51 (1:2,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). A control showing tubulin levels was performed using mouse anti-tubulin (1:12,000) antiserum.

    Techniques Used: Staining, Polymerase Chain Reaction, Generated, Amplification, Synthesized, Southern Blot, Purification, Agarose Gel Electrophoresis, SDS Page, Western Blot

    5) Product Images from "The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions"

    Article Title: The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006875

    TcRad51 immunolocalization in T . cruzi . A) WT, TcRAD51 +/- and TcRAD51 ox cells are shown before irradiation (non-irradiated) and 24 h after exposure to 500 Gy of gamma irradiation. DNA is shown stained with DAPI and TcRad51 was detected using anti-TcRad51 antibody raised in mouse (diluted 1:2,000) and visualized with Alexa 555 conjugated goat-derived anti-mouse IgG secondary (diluted 1:5,000); black bar: 5 μm. Cells are also shown in phase contrast but at a lower magnification (white bar: 2 μm). A graphical representation of fluorescence intensities is shown alongside the images. Vertical bars indicate standard deviation, asterisks represent statistically significant differences (*** p
    Figure Legend Snippet: TcRad51 immunolocalization in T . cruzi . A) WT, TcRAD51 +/- and TcRAD51 ox cells are shown before irradiation (non-irradiated) and 24 h after exposure to 500 Gy of gamma irradiation. DNA is shown stained with DAPI and TcRad51 was detected using anti-TcRad51 antibody raised in mouse (diluted 1:2,000) and visualized with Alexa 555 conjugated goat-derived anti-mouse IgG secondary (diluted 1:5,000); black bar: 5 μm. Cells are also shown in phase contrast but at a lower magnification (white bar: 2 μm). A graphical representation of fluorescence intensities is shown alongside the images. Vertical bars indicate standard deviation, asterisks represent statistically significant differences (*** p

    Techniques Used: Irradiation, Staining, Derivative Assay, Fluorescence, Standard Deviation

    T . cruzi response to treatment with DNA cross-linking agents. Sensitivity of WT, TcRAD51 ox and TcRAD51 -/+ cells to: (A) 0, 500 J/m 2 or 1.000 J/m 2 of UV-light, or (B) 25 μM, 50 μM or 75 μM cisplatin. Parasites were counted 48 h after treatment. Numbers are represented as a percentage of untreated cells. Values represent the mean of triplicates. Error bars indicate standard deviations.
    Figure Legend Snippet: T . cruzi response to treatment with DNA cross-linking agents. Sensitivity of WT, TcRAD51 ox and TcRAD51 -/+ cells to: (A) 0, 500 J/m 2 or 1.000 J/m 2 of UV-light, or (B) 25 μM, 50 μM or 75 μM cisplatin. Parasites were counted 48 h after treatment. Numbers are represented as a percentage of untreated cells. Values represent the mean of triplicates. Error bars indicate standard deviations.

    Techniques Used:

    Growth of T . cruzi following DNA damage treatment. A) The sensitivity of WT and TcRAD51 -/+ cells to gamma radiation was determined. Parasites were exposed to 0 Gy or 500 Gy of gamma irradiation, the number of parasites was determined every two days for 28 d. Values represent the mean of triplicates and error bars indicate standard deviations. B) Chromosomal profile from different T . cruzi (WT, TcRAD51 ox, and TcRAD51 +/- ) samples after treatment with gamma radiation (500 Gy). Chromosomal bands from different T . cruzi samples were separated by PFGE and stained using ethidium bromide. Whole chromosomes from Saccharomyces cerevisiae were used as molecular weight markers. (C) T . cruzi growth curve after treatment with 20 mM HU. Arrow indicates the point when the drug was removed. (D) T . cruzi growth curve after treatment with 1.5 mM MMS. The number of cells was determined every 24 h by vital staining. Curves shown are an example of three independent experiments that were performed in triplicate. Error bars represent standard deviations. (E) Detection of H2A levels in epimastigotes treated with 20 mM HU. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot using anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%). (F) Detection of H2A levels in epimastigotes treated with 1.5 mM MMS. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot with anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%).
    Figure Legend Snippet: Growth of T . cruzi following DNA damage treatment. A) The sensitivity of WT and TcRAD51 -/+ cells to gamma radiation was determined. Parasites were exposed to 0 Gy or 500 Gy of gamma irradiation, the number of parasites was determined every two days for 28 d. Values represent the mean of triplicates and error bars indicate standard deviations. B) Chromosomal profile from different T . cruzi (WT, TcRAD51 ox, and TcRAD51 +/- ) samples after treatment with gamma radiation (500 Gy). Chromosomal bands from different T . cruzi samples were separated by PFGE and stained using ethidium bromide. Whole chromosomes from Saccharomyces cerevisiae were used as molecular weight markers. (C) T . cruzi growth curve after treatment with 20 mM HU. Arrow indicates the point when the drug was removed. (D) T . cruzi growth curve after treatment with 1.5 mM MMS. The number of cells was determined every 24 h by vital staining. Curves shown are an example of three independent experiments that were performed in triplicate. Error bars represent standard deviations. (E) Detection of H2A levels in epimastigotes treated with 20 mM HU. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot using anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%). (F) Detection of H2A levels in epimastigotes treated with 1.5 mM MMS. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot with anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%).

    Techniques Used: Irradiation, Staining, Molecular Weight, SDS Page, Western Blot

    Generation of TcRAD51 -/+ knockouts mutants and TcRad51 protein levels. (A) Ethidium bromide stained gel showing PCR products generated with primers represented indicated in the schematic representation. PCR was performed using genomic DNA of WT and TcRAD51 +/- epimastigotes. Upper panel: schematic representation of RAD51 and hygromycin allele detection. Note that the amplicon is only synthesized if the pair of primers simultaneously hybridizes inside and outside the deletion cassette. The figure is not on scale. (B) Schematic representation of the Southern blot analysis showing BglI sites and the probes used (C) Left panel: Purified HYGB and RAD51 fragments generated by PCR-amplification. Right panel: Southern blot analysis of BglI- and BglI-digested genomic DNA from WT and TcRAD51 +/- cells and probed with HYGB and RAD51 fragments. Ethidium bromide-stained agarose gel showing the digestion products that were further analyzed by Southern blotting. (D) Detection of TcRad51 levels in epimastigotes protein extracts from WT, TcRAD51 ox, and TcRAD51 -/+ cells. Cellular lysates were separated by SDS-PAGE, and proteins were detected by Western blot with anti-TcRad51 (1:2,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). A control showing tubulin levels was performed using mouse anti-tubulin (1:12,000) antiserum.
    Figure Legend Snippet: Generation of TcRAD51 -/+ knockouts mutants and TcRad51 protein levels. (A) Ethidium bromide stained gel showing PCR products generated with primers represented indicated in the schematic representation. PCR was performed using genomic DNA of WT and TcRAD51 +/- epimastigotes. Upper panel: schematic representation of RAD51 and hygromycin allele detection. Note that the amplicon is only synthesized if the pair of primers simultaneously hybridizes inside and outside the deletion cassette. The figure is not on scale. (B) Schematic representation of the Southern blot analysis showing BglI sites and the probes used (C) Left panel: Purified HYGB and RAD51 fragments generated by PCR-amplification. Right panel: Southern blot analysis of BglI- and BglI-digested genomic DNA from WT and TcRAD51 +/- cells and probed with HYGB and RAD51 fragments. Ethidium bromide-stained agarose gel showing the digestion products that were further analyzed by Southern blotting. (D) Detection of TcRad51 levels in epimastigotes protein extracts from WT, TcRAD51 ox, and TcRAD51 -/+ cells. Cellular lysates were separated by SDS-PAGE, and proteins were detected by Western blot with anti-TcRad51 (1:2,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). A control showing tubulin levels was performed using mouse anti-tubulin (1:12,000) antiserum.

    Techniques Used: Staining, Polymerase Chain Reaction, Generated, Amplification, Synthesized, Southern Blot, Purification, Agarose Gel Electrophoresis, SDS Page, Western Blot

    6) Product Images from "Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens"

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02224

    Detection limits of IS6110 and senX3 PCR assays. Curves determined by probit analysis (95% probability detection). (A) IS6110 and senX3 PCR using M. tuberculosis mc 2 7000 DNA. LOD were estimated at 2.03 log 10 (107 copies/ml) and 2.89 log 10 (741 copies/ml), respectively. (B) The detection limit of M. bovis BCG was 2.40 log 10 and 2.90 log 10 for IS6110 and senX3 PCR assays, respectively. (C) IS6110 PCR detection limits using two or three sets of primers compared to a single set of primers based on M. tuberculosis mc 2 7000 strain were estimated at 2.1, 2.2, 2.3, 2.35, 2.4 log 10, respectively for ISP, Triplex, ISP+ISM, ISP+ISL, and ISM+ISL.
    Figure Legend Snippet: Detection limits of IS6110 and senX3 PCR assays. Curves determined by probit analysis (95% probability detection). (A) IS6110 and senX3 PCR using M. tuberculosis mc 2 7000 DNA. LOD were estimated at 2.03 log 10 (107 copies/ml) and 2.89 log 10 (741 copies/ml), respectively. (B) The detection limit of M. bovis BCG was 2.40 log 10 and 2.90 log 10 for IS6110 and senX3 PCR assays, respectively. (C) IS6110 PCR detection limits using two or three sets of primers compared to a single set of primers based on M. tuberculosis mc 2 7000 strain were estimated at 2.1, 2.2, 2.3, 2.35, 2.4 log 10, respectively for ISP, Triplex, ISP+ISM, ISP+ISL, and ISM+ISL.

    Techniques Used: Polymerase Chain Reaction

    Efficacy of MTB lysis using six different lysis methods combined with the Chelex ® resin extraction. Each column represents average DNA copy number per microliter obtained in five independent experiment with three replicate reactions.
    Figure Legend Snippet: Efficacy of MTB lysis using six different lysis methods combined with the Chelex ® resin extraction. Each column represents average DNA copy number per microliter obtained in five independent experiment with three replicate reactions.

    Techniques Used: Lysis

    Bland–Altman bias plots for two different quantitative MTBC DNA real-time PCR assays. Five serial dilutions of M. bovis BCG (A) and M. tuberculosis mc 2 7000 (B) strain were tested for MTBC DNA quantification by IS6110 and senX3 PCR assays. The mean bias was determined to be 4.026 and 7.455-cycle threshold for M. bovis BCG and M. tuberculosis mc 2 7000, respectively.
    Figure Legend Snippet: Bland–Altman bias plots for two different quantitative MTBC DNA real-time PCR assays. Five serial dilutions of M. bovis BCG (A) and M. tuberculosis mc 2 7000 (B) strain were tested for MTBC DNA quantification by IS6110 and senX3 PCR assays. The mean bias was determined to be 4.026 and 7.455-cycle threshold for M. bovis BCG and M. tuberculosis mc 2 7000, respectively.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Comparison of DNA extraction protocols in spiked sputum samples. The M. tuberculosis mc 2 7000 stock suspension was diluted and used to spike negative sputum samples. Box plots with C T median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ® method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.
    Figure Legend Snippet: Comparison of DNA extraction protocols in spiked sputum samples. The M. tuberculosis mc 2 7000 stock suspension was diluted and used to spike negative sputum samples. Box plots with C T median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ® method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.

    Techniques Used: DNA Extraction, Sonication

    MTB DNA concentration in culture positive samples stratified according to smear microscopy results using the IS6110 PCR (A) . Comparison of real-time PCR IS6110 assay and Xpert MTB/RIF on 40 clinical sputum samples. Positive threshold of the PCR assay is indicated by the dotted line (B) .
    Figure Legend Snippet: MTB DNA concentration in culture positive samples stratified according to smear microscopy results using the IS6110 PCR (A) . Comparison of real-time PCR IS6110 assay and Xpert MTB/RIF on 40 clinical sputum samples. Positive threshold of the PCR assay is indicated by the dotted line (B) .

    Techniques Used: Concentration Assay, Microscopy, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    7) Product Images from "C1q/ TNF‐related peptide 8 ( CTRP8) promotes temozolomide resistance in human glioblastoma"

    Article Title: C1q/ TNF‐related peptide 8 ( CTRP8) promotes temozolomide resistance in human glioblastoma

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12349

    CTRP 8 reduces the number of AP sites and enhances BER . We quantified AP sites in genomic DNA to determine the cause of TMZ ‐induced DNA damage. The endogenous level of AP sites was about 12–15 sites/10 5 bp in our patient GBM ‐1 model (A). TMZ alone triggered a marked upregulation of AP sites in patient GBM ‐1 cells (A). A significant reduction in the number of AP sites comparable to levels detected in untreated control cells was observed upon pretreatment of patient GBM ‐1 cells with CTRP 8 followed by TMZ exposure (A). CTRP 8 treatment alone did not alter the number of AP sites (A). Western blot analysis of important BER proteins showed that CTRP 8 (100 ng·mL −1 ) induced the specific and exclusive upregulation of N‐methylpurine DNA glycosylase ( MPG ) protein, a key factor in initiating BER , in patient GBM ‐1 (B) and U87 MG (C). Importantly, si RXFP 1 KD and STAT 3 inhibition abolished this CTRP 8 mediated increase in MPG protein (B–G) as shown for patient GBM ‐1 (B, D, F) and U87 MG (C, E, G). The increase in MPG protein coincided with a significant upregulation of MPG transcripts upon CTRP 8 treatment as revealed by QPCR analysis in patient GBM ‐1 (H) and U87 MG (I). We used an MPG ‐specific molecular beacon activity assay on U87 MG nuclear lysates which utilizes a specific MPG deoxyribonucleotide probe with a fluorophore (6‐ FAM ) attached at the 5′‐end and a quencher (3Dab) at the 3′‐end (Svilar et al ., 2012 ) (J). This MPG oligo probe contains a deoxyinosine ( dI ) base as MPG recognition site. The exclusive MPG cleavage at this site releases a 6‐bp DNA fragment with attached 5′‐fluorophore which dissociates from its quencher to generate a fluorescence signal which is proportional to MPG activity and can be quantified by qPCR (J). An identical control oligo lacking the dI base and, thus, is not cleaved by MPG was used as control. Quantification of fluorescence intensities reflecting MPG activities measured at 60, 120, and 180 min is shown (K). Endogenous MPG exclusively cleaved the MPG probe, and CTRP 8 treatment caused a further significant increase in MPG activity which was absent in the presence of S3I‐201 (K). Quantitative analysis from three independent experiments (two‐way ANOVA ; data are shown as mean ± SD ; * P
    Figure Legend Snippet: CTRP 8 reduces the number of AP sites and enhances BER . We quantified AP sites in genomic DNA to determine the cause of TMZ ‐induced DNA damage. The endogenous level of AP sites was about 12–15 sites/10 5 bp in our patient GBM ‐1 model (A). TMZ alone triggered a marked upregulation of AP sites in patient GBM ‐1 cells (A). A significant reduction in the number of AP sites comparable to levels detected in untreated control cells was observed upon pretreatment of patient GBM ‐1 cells with CTRP 8 followed by TMZ exposure (A). CTRP 8 treatment alone did not alter the number of AP sites (A). Western blot analysis of important BER proteins showed that CTRP 8 (100 ng·mL −1 ) induced the specific and exclusive upregulation of N‐methylpurine DNA glycosylase ( MPG ) protein, a key factor in initiating BER , in patient GBM ‐1 (B) and U87 MG (C). Importantly, si RXFP 1 KD and STAT 3 inhibition abolished this CTRP 8 mediated increase in MPG protein (B–G) as shown for patient GBM ‐1 (B, D, F) and U87 MG (C, E, G). The increase in MPG protein coincided with a significant upregulation of MPG transcripts upon CTRP 8 treatment as revealed by QPCR analysis in patient GBM ‐1 (H) and U87 MG (I). We used an MPG ‐specific molecular beacon activity assay on U87 MG nuclear lysates which utilizes a specific MPG deoxyribonucleotide probe with a fluorophore (6‐ FAM ) attached at the 5′‐end and a quencher (3Dab) at the 3′‐end (Svilar et al ., 2012 ) (J). This MPG oligo probe contains a deoxyinosine ( dI ) base as MPG recognition site. The exclusive MPG cleavage at this site releases a 6‐bp DNA fragment with attached 5′‐fluorophore which dissociates from its quencher to generate a fluorescence signal which is proportional to MPG activity and can be quantified by qPCR (J). An identical control oligo lacking the dI base and, thus, is not cleaved by MPG was used as control. Quantification of fluorescence intensities reflecting MPG activities measured at 60, 120, and 180 min is shown (K). Endogenous MPG exclusively cleaved the MPG probe, and CTRP 8 treatment caused a further significant increase in MPG activity which was absent in the presence of S3I‐201 (K). Quantitative analysis from three independent experiments (two‐way ANOVA ; data are shown as mean ± SD ; * P

    Techniques Used: Western Blot, Inhibition, Real-time Polymerase Chain Reaction, Activity Assay, Fluorescence

    Schematic model of the CTRP 8‐ RXFP 1‐ STAT 3 signaling axis in human GBM . We propose a model in which the interaction of CTRP 8 with membrane‐anchored RXFP 1 triggers a pSTAT 3 signaling cascade in human GBM . STAT 3 activation enhances MPG ‐dependent BER , thereby reducing DNA damage and promoting GBM survival. The latter includes the upregulation of anti‐apoptotic STAT 3 targets Bcl‐2 and Bcl‐ XL . Collectively, this establishes the CTRP 8‐ RXFP 1‐ STAT 3 cascade as a novel oncogenic signaling pathway that promotes TMZ chemoresistance in human high‐grade glioma.
    Figure Legend Snippet: Schematic model of the CTRP 8‐ RXFP 1‐ STAT 3 signaling axis in human GBM . We propose a model in which the interaction of CTRP 8 with membrane‐anchored RXFP 1 triggers a pSTAT 3 signaling cascade in human GBM . STAT 3 activation enhances MPG ‐dependent BER , thereby reducing DNA damage and promoting GBM survival. The latter includes the upregulation of anti‐apoptotic STAT 3 targets Bcl‐2 and Bcl‐ XL . Collectively, this establishes the CTRP 8‐ RXFP 1‐ STAT 3 cascade as a novel oncogenic signaling pathway that promotes TMZ chemoresistance in human high‐grade glioma.

    Techniques Used: Activation Assay

    CTRP 8 attenuates TMZ ‐induced DNA damage. γH2 AX , a marker of double‐strand (ds) DNA breaks, was detected by immunofluorescence in patient GBM (A). Treatment with TMZ (1.5 m m ) resulted in a significant increase in γH2 AX foci (red) in DAPI ‐stained nuclei (blue) compared to medium controls (A). Pretreatment for 24 h with CTRP 8 (100 ng·mL −1 ) caused a marked reduction in γH2 AX foci upon exposure to TMZ compared to patient GBM ‐1 cells treated with TMZ alone (A). This CTRP 8‐mediated DNA protective effect was abolished upon si RXFP 1‐1 KD (A). The results of the quantification of fluorescence intensity of γH2 AX foci for 100 nuclei per treatment group are shown (B). Upon TMZ treatment, western blot analysis (C–F) revealed a marked reduction in γH2 AX protein in the presence of CTRP 8 in patient GBM ‐1 (C, E) and U87 MG (D, F). This DNA protective effect of CTRP 8 was blocked upon si RXFP 1‐1 treatment in patient GBM ‐1 (C) and U87 MG (D). Similar results were obtained with STAT 3 inhibitor S3I‐201 in patient GBM ‐1 (E) and U87 MG (F). β‐Actin served as loading control. Representative western blot images are shown. Densitometry of western blot signal for γH2 AX in patient GBM ‐1 (G) and U87 MG (H) was normalized to β‐actin. We employed Comet assay to quantify DNA fragmentation as determined by olive tail moment ( OTM ) in individual human GBM ‐1 cells (I, J). Pretreatment for 24 h with CTRP 8 (100 ng·mL −1 ) prior to TMZ exposure (1.5 m m ) resulted in decreased OTM , as shown in representative agarose gel images of comets from patient GBM ‐1 cells (I, J). The OTM was determined as an index of both comet tail length and the amount of DNA in tail as quantified by SYBR green fluorescence dye (L). Quantitative analysis of OTM from 50 cells for each treatment group revealed that CTRP 8 caused a marked reduction in ds DNA breaks. This protective CTRP 8 function was lost upon treatment with S3I‐201 in patient GBM ‐1 (K) and U87 MG (M). CTRP 8 and S3I‐201 alone failed to cause ds DNA breaks and resulted in negligible OTM (K, M). Quantitative analysis from three independent experiments (two‐way ANOVA ; data are shown as mean ± SD ; **** P
    Figure Legend Snippet: CTRP 8 attenuates TMZ ‐induced DNA damage. γH2 AX , a marker of double‐strand (ds) DNA breaks, was detected by immunofluorescence in patient GBM (A). Treatment with TMZ (1.5 m m ) resulted in a significant increase in γH2 AX foci (red) in DAPI ‐stained nuclei (blue) compared to medium controls (A). Pretreatment for 24 h with CTRP 8 (100 ng·mL −1 ) caused a marked reduction in γH2 AX foci upon exposure to TMZ compared to patient GBM ‐1 cells treated with TMZ alone (A). This CTRP 8‐mediated DNA protective effect was abolished upon si RXFP 1‐1 KD (A). The results of the quantification of fluorescence intensity of γH2 AX foci for 100 nuclei per treatment group are shown (B). Upon TMZ treatment, western blot analysis (C–F) revealed a marked reduction in γH2 AX protein in the presence of CTRP 8 in patient GBM ‐1 (C, E) and U87 MG (D, F). This DNA protective effect of CTRP 8 was blocked upon si RXFP 1‐1 treatment in patient GBM ‐1 (C) and U87 MG (D). Similar results were obtained with STAT 3 inhibitor S3I‐201 in patient GBM ‐1 (E) and U87 MG (F). β‐Actin served as loading control. Representative western blot images are shown. Densitometry of western blot signal for γH2 AX in patient GBM ‐1 (G) and U87 MG (H) was normalized to β‐actin. We employed Comet assay to quantify DNA fragmentation as determined by olive tail moment ( OTM ) in individual human GBM ‐1 cells (I, J). Pretreatment for 24 h with CTRP 8 (100 ng·mL −1 ) prior to TMZ exposure (1.5 m m ) resulted in decreased OTM , as shown in representative agarose gel images of comets from patient GBM ‐1 cells (I, J). The OTM was determined as an index of both comet tail length and the amount of DNA in tail as quantified by SYBR green fluorescence dye (L). Quantitative analysis of OTM from 50 cells for each treatment group revealed that CTRP 8 caused a marked reduction in ds DNA breaks. This protective CTRP 8 function was lost upon treatment with S3I‐201 in patient GBM ‐1 (K) and U87 MG (M). CTRP 8 and S3I‐201 alone failed to cause ds DNA breaks and resulted in negligible OTM (K, M). Quantitative analysis from three independent experiments (two‐way ANOVA ; data are shown as mean ± SD ; **** P

    Techniques Used: Marker, Immunofluorescence, Staining, Fluorescence, Western Blot, Single Cell Gel Electrophoresis, Agarose Gel Electrophoresis, SYBR Green Assay

    8) Product Images from "Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery"

    Article Title: Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

    Journal: Genetic Vaccines and Therapy

    doi: 10.1186/1479-0556-4-1

    Identification of plasmid DNA rescued. Nature of plasmid DNA obtained after transformation of cellular DNA from tissues of mice 2 days after i.m. immunization with pcDNA3-Hsp65. Escherichia coli DH5-α was transformed with 1 μg of total DNA from tissues of mice previously immunized with pcDNA3-Hsp65. Plasmid DNA was recovered from ampicillin-resistant colonies. (A) Agarose gel showing plasmid DNA digested overnight with Hind III and Xba I: wild-type pcDNA3-Hsp65 (lane a); plasmid DNA recovered from muscle (lane b); plasmid DNA from bone marrow (lane c) plasmid DNA from liver (lane d); plasmid DNA from spleen (lane e). (B) PCR analysis of rescued plasmid using HSP65 primers: Wild type pcDNA3-Hsp65 (lane a); plasmid rescued from muscle of immunized mice (lane b). The mobility of DNA size standards (λ DNA cut with Hind III) are shown on the left. (C) Identification of nucleotide sequence of plasmid DNA rescued from muscle. Sequence analyses were performed using the blastn program from BLAST.
    Figure Legend Snippet: Identification of plasmid DNA rescued. Nature of plasmid DNA obtained after transformation of cellular DNA from tissues of mice 2 days after i.m. immunization with pcDNA3-Hsp65. Escherichia coli DH5-α was transformed with 1 μg of total DNA from tissues of mice previously immunized with pcDNA3-Hsp65. Plasmid DNA was recovered from ampicillin-resistant colonies. (A) Agarose gel showing plasmid DNA digested overnight with Hind III and Xba I: wild-type pcDNA3-Hsp65 (lane a); plasmid DNA recovered from muscle (lane b); plasmid DNA from bone marrow (lane c) plasmid DNA from liver (lane d); plasmid DNA from spleen (lane e). (B) PCR analysis of rescued plasmid using HSP65 primers: Wild type pcDNA3-Hsp65 (lane a); plasmid rescued from muscle of immunized mice (lane b). The mobility of DNA size standards (λ DNA cut with Hind III) are shown on the left. (C) Identification of nucleotide sequence of plasmid DNA rescued from muscle. Sequence analyses were performed using the blastn program from BLAST.

    Techniques Used: Plasmid Preparation, Transformation Assay, Mouse Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing

    Persistence of DNA adenine methylase site methylations (dam) of pcDNA3-HSP65 in muscle at 6 months after immunization . (A) Approximately 1 μg cellular DNA obtained from muscle of immunized mouse were digested with Nde I and Dpn I (lane a), Nde I and Mbo I (lane b), or with Nde I alone (lane c) and amplified by PCR using Hsp65 primers. The samples were submitted to electrophoresis on a 1% agarose gel (B) The positive control was done using E.coli DNA digested with Dpn I, Mbo I or non-digested to show the dam methylation pattern. The mobility of DNA size standards (l DNA cut with Hind III) are shown on the left.
    Figure Legend Snippet: Persistence of DNA adenine methylase site methylations (dam) of pcDNA3-HSP65 in muscle at 6 months after immunization . (A) Approximately 1 μg cellular DNA obtained from muscle of immunized mouse were digested with Nde I and Dpn I (lane a), Nde I and Mbo I (lane b), or with Nde I alone (lane c) and amplified by PCR using Hsp65 primers. The samples were submitted to electrophoresis on a 1% agarose gel (B) The positive control was done using E.coli DNA digested with Dpn I, Mbo I or non-digested to show the dam methylation pattern. The mobility of DNA size standards (l DNA cut with Hind III) are shown on the left.

    Techniques Used: Amplification, Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Positive Control, Methylation

    Analysis of the pcDNA3-Hsp65 genome integration . Samples of liver tissue from mice immunized with 100 μg of pcDNA3-HSP65 (lanes a and b) and from nonimmunized mice (lanes c and d) (negative control) were submitted to Southern blot after Nde I digestion. Lanes e and f correspond to wild-type plasmid digested with Nde I or undigested, respectively. The bands were detected using pcDNA3 labeled with chemiluminescent reagent. The multiple forms of plasmid DNA are indicated in the figure. The samples were loaded in a same gel and the lanes not used were removed.
    Figure Legend Snippet: Analysis of the pcDNA3-Hsp65 genome integration . Samples of liver tissue from mice immunized with 100 μg of pcDNA3-HSP65 (lanes a and b) and from nonimmunized mice (lanes c and d) (negative control) were submitted to Southern blot after Nde I digestion. Lanes e and f correspond to wild-type plasmid digested with Nde I or undigested, respectively. The bands were detected using pcDNA3 labeled with chemiluminescent reagent. The multiple forms of plasmid DNA are indicated in the figure. The samples were loaded in a same gel and the lanes not used were removed.

    Techniques Used: Mouse Assay, Negative Control, Southern Blot, Plasmid Preparation, Labeling

    9) Product Images from "TALEN mediated somatic mutagenesis in murine models of cancer"

    Article Title: TALEN mediated somatic mutagenesis in murine models of cancer

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-14-0529

    Ape TALENs are effective and efficient in vitro and in xenografts (A) Design of Apc GoldyTALEN. The target region in exon 9 contains a PvuII cut site. (B) Protein expression of Apc TALENs in H2.35 cells. H2.35 cells were transfected with GoldyTALEN vector, single TALENs, or the TALEN pair. Cells were harvested and subjected to western blot 48 hours after transfection. (C) Apc TALEN efficiency in H2.35 cells. Genomic DNA was PCR amplified for the region targeted by TALENs and digested by PvuII. TALEN efficiency is determined by the ratio of uncut band vs. total bands. (D) IF staining in H2.35 cells for β-catenin . Cells were fixed after 72 hours of transfection and then stained for β-catenin (Green). Cytoplasmic localization of β-catenin (arrow) is seen in cells treated with the TALEN pair. (E) Western blot for Apc in H2.35 cell clones and the sequencing data of Apc mutant clone #29. The H2.35 parental cells and WT clone #25 express full length Apc , and mutant clone #29 has no full length Apc , as well as the predicted truncated Apc . (F) Growth curve of the H2.35 xenografts subcutaneously transplanted into nude mice.
    Figure Legend Snippet: Ape TALENs are effective and efficient in vitro and in xenografts (A) Design of Apc GoldyTALEN. The target region in exon 9 contains a PvuII cut site. (B) Protein expression of Apc TALENs in H2.35 cells. H2.35 cells were transfected with GoldyTALEN vector, single TALENs, or the TALEN pair. Cells were harvested and subjected to western blot 48 hours after transfection. (C) Apc TALEN efficiency in H2.35 cells. Genomic DNA was PCR amplified for the region targeted by TALENs and digested by PvuII. TALEN efficiency is determined by the ratio of uncut band vs. total bands. (D) IF staining in H2.35 cells for β-catenin . Cells were fixed after 72 hours of transfection and then stained for β-catenin (Green). Cytoplasmic localization of β-catenin (arrow) is seen in cells treated with the TALEN pair. (E) Western blot for Apc in H2.35 cell clones and the sequencing data of Apc mutant clone #29. The H2.35 parental cells and WT clone #25 express full length Apc , and mutant clone #29 has no full length Apc , as well as the predicted truncated Apc . (F) Growth curve of the H2.35 xenografts subcutaneously transplanted into nude mice.

    Techniques Used: TALENs, In Vitro, Expressing, Transfection, Plasmid Preparation, Western Blot, Polymerase Chain Reaction, Amplification, Staining, Clone Assay, Sequencing, Mutagenesis, Mouse Assay

    β-catenin TALENs are effective and efficient in vitro (A) Design of β-catenin ( β-cat ) GoldyTALEN. The target region contains β-catenin codon 32 and 33 and an XmnI cut site between the two TALENs. (B) Protein expression of β-catenin TALENs in H2.35 cells. H2.35 cells were transfected with GoldyTALEN control vector, single TALENs, or TALEN pairs. Cells were harvested and subjected to western blot 48 hours after transfection. AcV5 is an epitope tag fused to the TALENs. (C) β-catenin TALEN efficiency in H2.35 cells. Genomic DNA was PCR amplified for the region targeted by TALENs and digested by XmnI. The uncut band in A lane is mutated DNA. TALEN efficiency is determined by the ratio of uncut band vs. total bands. (D) IF staining in H2.35 cells for β-catenin . Cells were fixed 72 hours after transfection and stained for β-catenin (Green). Cytoplasmic localization of β-catenin (arrow) is seen in cells treated with the TALEN pair.
    Figure Legend Snippet: β-catenin TALENs are effective and efficient in vitro (A) Design of β-catenin ( β-cat ) GoldyTALEN. The target region contains β-catenin codon 32 and 33 and an XmnI cut site between the two TALENs. (B) Protein expression of β-catenin TALENs in H2.35 cells. H2.35 cells were transfected with GoldyTALEN control vector, single TALENs, or TALEN pairs. Cells were harvested and subjected to western blot 48 hours after transfection. AcV5 is an epitope tag fused to the TALENs. (C) β-catenin TALEN efficiency in H2.35 cells. Genomic DNA was PCR amplified for the region targeted by TALENs and digested by XmnI. The uncut band in A lane is mutated DNA. TALEN efficiency is determined by the ratio of uncut band vs. total bands. (D) IF staining in H2.35 cells for β-catenin . Cells were fixed 72 hours after transfection and stained for β-catenin (Green). Cytoplasmic localization of β-catenin (arrow) is seen in cells treated with the TALEN pair.

    Techniques Used: TALENs, In Vitro, Expressing, Transfection, Plasmid Preparation, Western Blot, Polymerase Chain Reaction, Amplification, Staining

    Hepatocyte polyploidy protects the liver from TALEN mediated tumor suppressor loss (A) Genotyping of mouse liver treated with Apc TALENs by HDT. Mice underwent HDT with GoldyTALEN vector (20ug), Apc single TALENs (20ug), or TALEN pair (10ug of each TALEN) at 6 weeks of age, then weekly × 4 (left panel) or × 2 (right panel). (B) FACS showing DNA content distribution, as stained by PI/RNAse, of primary hepatocytes in a 1 month old C3H mouse. The right two panels are liver IF images showing a mononucleated and a binucleated hepatocyte. β-catenin (purple) stains the membrane. (C) The left panel shows the DNA content distribution for H2.35 cells, which were stained by Hoechst 33342 and sorted into 2c and 4c populations. Representative karyotypes of the sorted cells are shown. (D) Ploidy distribution within the sorted populations. 50 cells were counted for each population. (E) Experiment assessing the efficiency of TALEN induced Apc LOH in diploid vs. tetroploid cells. H2.35 cells were transfected with TALENs and a GFP plasmid. GFP+ cells were sorted and separated by ploidy and plated as single cell clones. Genotyping was performed on individual clones. (F) DNA content distribution (Hoechst 33342) of the H2.35 cells transfected with Apc TALENs. (G) Genotyping of the H2.35 cell single clones described in (E) and (F). The red rectangles highlight homozygous clones. The experiment was performed four times and the overall percentage of homozygous clones is shown on the right.
    Figure Legend Snippet: Hepatocyte polyploidy protects the liver from TALEN mediated tumor suppressor loss (A) Genotyping of mouse liver treated with Apc TALENs by HDT. Mice underwent HDT with GoldyTALEN vector (20ug), Apc single TALENs (20ug), or TALEN pair (10ug of each TALEN) at 6 weeks of age, then weekly × 4 (left panel) or × 2 (right panel). (B) FACS showing DNA content distribution, as stained by PI/RNAse, of primary hepatocytes in a 1 month old C3H mouse. The right two panels are liver IF images showing a mononucleated and a binucleated hepatocyte. β-catenin (purple) stains the membrane. (C) The left panel shows the DNA content distribution for H2.35 cells, which were stained by Hoechst 33342 and sorted into 2c and 4c populations. Representative karyotypes of the sorted cells are shown. (D) Ploidy distribution within the sorted populations. 50 cells were counted for each population. (E) Experiment assessing the efficiency of TALEN induced Apc LOH in diploid vs. tetroploid cells. H2.35 cells were transfected with TALENs and a GFP plasmid. GFP+ cells were sorted and separated by ploidy and plated as single cell clones. Genotyping was performed on individual clones. (F) DNA content distribution (Hoechst 33342) of the H2.35 cells transfected with Apc TALENs. (G) Genotyping of the H2.35 cell single clones described in (E) and (F). The red rectangles highlight homozygous clones. The experiment was performed four times and the overall percentage of homozygous clones is shown on the right.

    Techniques Used: TALENs, Mouse Assay, Plasmid Preparation, FACS, Staining, Transfection, Clone Assay

    10) Product Images from "Controlled insertional mutagenesis using a LINE-1 (ORFeus) gene-trap mouse model"

    Article Title: Controlled insertional mutagenesis using a LINE-1 (ORFeus) gene-trap mouse model

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1302504110

    Confirmation of tet- ORFeus expression and somatic retrotransposition in vivo. ( A ) Genotyping PCR (ORF2 from tet- ORFeus and CMVrtTA) and gene-trap splicing assay (GT3-1.2) with genomic DNA isolated from tail tissue in progeny of heterozygous tet- ORFeus and CMVrtTA animals. Line 058 mice were treated with 0.1 mg/mL doxycycline during embryogenesis. Blue arrows below the gel images indicate double-transgenic progeny. NTC, no template control. ( B and C ) Quantitative real-time RT-PCR analysis of CMVrtTA ( B ) and ORF2 ( C ) expression in a panel of tissues from the double-transgenic line 058 animal treated with 0.1 mg/mL doxycycline. Bar graphs represent mean ORF2 and CMVrtTA expression relative to actin and normalized to the liver sample for each amplicon, respectively. Error bars represent SDs from three independent measurements. ( D ) Inverse PCR retrotransposition assay with genomic DNA isolated from a double-transgenic animal (line 058) treated in utero with 0.1 mg/mL doxycycline. For each sample, three independent iPCR reactions were performed using the same ligation mixture. Genomic DNA isolated from liver of the CAG- ORFeus transgenic mouse line (line 210 liver) serves as a reference. ( E ) Inverse PCR assay with tail gDNA isolated from four littermates (line 058) demonstrating that retrotransposition activity is specific to double-transgenic animals treated with doxycycline. Genotypes are labeled above gel image. For each tail gDNA sample, four independent reactions were performed with the same ligation mix.
    Figure Legend Snippet: Confirmation of tet- ORFeus expression and somatic retrotransposition in vivo. ( A ) Genotyping PCR (ORF2 from tet- ORFeus and CMVrtTA) and gene-trap splicing assay (GT3-1.2) with genomic DNA isolated from tail tissue in progeny of heterozygous tet- ORFeus and CMVrtTA animals. Line 058 mice were treated with 0.1 mg/mL doxycycline during embryogenesis. Blue arrows below the gel images indicate double-transgenic progeny. NTC, no template control. ( B and C ) Quantitative real-time RT-PCR analysis of CMVrtTA ( B ) and ORF2 ( C ) expression in a panel of tissues from the double-transgenic line 058 animal treated with 0.1 mg/mL doxycycline. Bar graphs represent mean ORF2 and CMVrtTA expression relative to actin and normalized to the liver sample for each amplicon, respectively. Error bars represent SDs from three independent measurements. ( D ) Inverse PCR retrotransposition assay with genomic DNA isolated from a double-transgenic animal (line 058) treated in utero with 0.1 mg/mL doxycycline. For each sample, three independent iPCR reactions were performed using the same ligation mixture. Genomic DNA isolated from liver of the CAG- ORFeus transgenic mouse line (line 210 liver) serves as a reference. ( E ) Inverse PCR assay with tail gDNA isolated from four littermates (line 058) demonstrating that retrotransposition activity is specific to double-transgenic animals treated with doxycycline. Genotypes are labeled above gel image. For each tail gDNA sample, four independent reactions were performed with the same ligation mix.

    Techniques Used: Expressing, In Vivo, Polymerase Chain Reaction, Splicing Assay, Isolation, Mouse Assay, Transgenic Assay, Quantitative RT-PCR, Amplification, Inverse PCR, In Utero, Ligation, Activity Assay, Labeling

    Generation of a conditional LINE-1 retrotransposon. ( A ) Total RNA blot analysis demonstrating doxycycline (Dox)-regulated expression of tet- ORFeus in Tet-ON HeLa cells. ORF2 probe is derived from ORFeus template. ARPPo serves as a loading control. ( B ) Total RNA blot analysis in Tet-OFF HeLa cells. ( C ) RNA blot analysis of ORF2 expression in constructs from which the ORFeus transgene is driven by either the CAG promoter or the TRE promoter. ( D ) carrying the indicated donor ORFeus element in the presence or absence of doxycycline (Dox). The numbers in the first column represent the number of transposition events (i.e., the number of colonies per microgram input of DNA) normalized to the number obtained with pCAG- ORFeus . The numbers on the bottom indicate the number of puromycin-resistant cells plated.
    Figure Legend Snippet: Generation of a conditional LINE-1 retrotransposon. ( A ) Total RNA blot analysis demonstrating doxycycline (Dox)-regulated expression of tet- ORFeus in Tet-ON HeLa cells. ORF2 probe is derived from ORFeus template. ARPPo serves as a loading control. ( B ) Total RNA blot analysis in Tet-OFF HeLa cells. ( C ) RNA blot analysis of ORF2 expression in constructs from which the ORFeus transgene is driven by either the CAG promoter or the TRE promoter. ( D ) carrying the indicated donor ORFeus element in the presence or absence of doxycycline (Dox). The numbers in the first column represent the number of transposition events (i.e., the number of colonies per microgram input of DNA) normalized to the number obtained with pCAG- ORFeus . The numbers on the bottom indicate the number of puromycin-resistant cells plated.

    Techniques Used: Northern blot, Expressing, Derivative Assay, Construct

    Embryonic lethality and dose-dependent retrotransposition in mice with a high mutagenic burden of L1 insertions. ( A ) The percentage of each genotypic class was calculated with increasing doses of doxycycline. Four possible genotypes are expected with equal representation if there is no effect on viability (25% tet- ORFeus /+; CMVrtTA/+; 25% tet- ORFeus /+; +/+; 25% +/+; CMVrtTA/+; 25% +/+; +/+). Only the double-transgenic class (tet- ORFeus /+; CMVrtTA/+) was under-represented at high doses of doxycycline. The number of animals analyzed at each dose are as follows: untreated– n = 48; 0.1 mg/mL– n = 103; 0.25 mg/mL– n = 92; 0.5 mg/mL– n = 135; 1.0 mg/mL– n = 20; 2.0 mg/mL– n = 14. ( B ) Genotyping PCR (ORF2 and CMVrtTA) and gene-trap splicing assay (GT3-1.2) with genomic DNA isolated from E14.5 embryos. Animals were treated with either 0.1 or 0.5 mg/mL doxycycline beginning at conception. Numbers represent individual embryos. Asterisks below the gel image indicate double-transgenic embryos. HPRT serves as a control for equal loading of template gDNA. ( C ) iPCR assay with genomic DNA isolated from four different embryos. The genotype and doxycycline dose for each embryo are labeled above the gel image. For each embryo, six independent iPCR reactions were performed.
    Figure Legend Snippet: Embryonic lethality and dose-dependent retrotransposition in mice with a high mutagenic burden of L1 insertions. ( A ) The percentage of each genotypic class was calculated with increasing doses of doxycycline. Four possible genotypes are expected with equal representation if there is no effect on viability (25% tet- ORFeus /+; CMVrtTA/+; 25% tet- ORFeus /+; +/+; 25% +/+; CMVrtTA/+; 25% +/+; +/+). Only the double-transgenic class (tet- ORFeus /+; CMVrtTA/+) was under-represented at high doses of doxycycline. The number of animals analyzed at each dose are as follows: untreated– n = 48; 0.1 mg/mL– n = 103; 0.25 mg/mL– n = 92; 0.5 mg/mL– n = 135; 1.0 mg/mL– n = 20; 2.0 mg/mL– n = 14. ( B ) Genotyping PCR (ORF2 and CMVrtTA) and gene-trap splicing assay (GT3-1.2) with genomic DNA isolated from E14.5 embryos. Animals were treated with either 0.1 or 0.5 mg/mL doxycycline beginning at conception. Numbers represent individual embryos. Asterisks below the gel image indicate double-transgenic embryos. HPRT serves as a control for equal loading of template gDNA. ( C ) iPCR assay with genomic DNA isolated from four different embryos. The genotype and doxycycline dose for each embryo are labeled above the gel image. For each embryo, six independent iPCR reactions were performed.

    Techniques Used: Mouse Assay, Transgenic Assay, Polymerase Chain Reaction, Splicing Assay, Isolation, Labeling

    11) Product Images from "Gene number determination and genetic polymorphism of the gamma delta T cell co-receptor WC1 genes"

    Article Title: Gene number determination and genetic polymorphism of the gamma delta T cell co-receptor WC1 genes

    Journal: BMC Genetics

    doi: 10.1186/1471-2156-13-86

    Genomic DNA and cDNA evidence for the 13 WC1 genes in two bovine breeds. PCR analysis was conducted by using genomic DNA and cDNA derived from cattle of two different breeds (Belted Galloway, designed with prefixes of “Y_gDNA and Y_cDNA”, respectively; Holstein, designed with prefixes of “B_gDNA and B_cDNA”, respectively). Primer pairs for distinguishing thirteen WC1 domain 1s were thirteen WC1 specific primer sets. For each primer set, the identities of the amplified products were confirmed by DNA sequencing analysis.
    Figure Legend Snippet: Genomic DNA and cDNA evidence for the 13 WC1 genes in two bovine breeds. PCR analysis was conducted by using genomic DNA and cDNA derived from cattle of two different breeds (Belted Galloway, designed with prefixes of “Y_gDNA and Y_cDNA”, respectively; Holstein, designed with prefixes of “B_gDNA and B_cDNA”, respectively). Primer pairs for distinguishing thirteen WC1 domain 1s were thirteen WC1 specific primer sets. For each primer set, the identities of the amplified products were confirmed by DNA sequencing analysis.

    Techniques Used: Polymerase Chain Reaction, Derivative Assay, Amplification, DNA Sequencing

    Specific amplification of each WC1 Domain 1. (A) Primer specificities of thirteen WC1 specific primer sets. Confirmation of PCR amplification specificities by gel electrophoresis for thirteen WC1 specific primer sets ( WC1-1, WC1-3, WC1-4, WC1-5, WC1-6, WC1-7, WC1-9, WC1-10, WC1-11, WC1-12, WC1-13, WC1-nd1, and WC1-nd2 ) and the bovine IFNB primer pair. Plasmids containing thirteen WC1 domain 1 gene sequences were used as templates in PCR reactions. For each primer set, the identities of the amplified products were confirmed by DNA sequencing analysis. (B) Genomic DNA and cDNA evidence for Dominette. PCR analysis was conducted by using genomic DNA and cDNA derived from the reference animal Dominette of the Hereford breed (designed with prefixes of “D_gDNA and D_cDNA”, respectively). Primer pairs for amplification of thirteen WC1 SRCR domain 1 sequences with the specific primer sets tested in (A). For each primer set, the identities of the amplified products were confirmed by DNA sequencing analysis.
    Figure Legend Snippet: Specific amplification of each WC1 Domain 1. (A) Primer specificities of thirteen WC1 specific primer sets. Confirmation of PCR amplification specificities by gel electrophoresis for thirteen WC1 specific primer sets ( WC1-1, WC1-3, WC1-4, WC1-5, WC1-6, WC1-7, WC1-9, WC1-10, WC1-11, WC1-12, WC1-13, WC1-nd1, and WC1-nd2 ) and the bovine IFNB primer pair. Plasmids containing thirteen WC1 domain 1 gene sequences were used as templates in PCR reactions. For each primer set, the identities of the amplified products were confirmed by DNA sequencing analysis. (B) Genomic DNA and cDNA evidence for Dominette. PCR analysis was conducted by using genomic DNA and cDNA derived from the reference animal Dominette of the Hereford breed (designed with prefixes of “D_gDNA and D_cDNA”, respectively). Primer pairs for amplification of thirteen WC1 SRCR domain 1 sequences with the specific primer sets tested in (A). For each primer set, the identities of the amplified products were confirmed by DNA sequencing analysis.

    Techniques Used: Amplification, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, DNA Sequencing, Derivative Assay

    Amplification of full-length transcripts for WC1s. (A) Primers designed for complete coding sequences of all thirteen WC1 genes. Schematic representations of the molecular forms of archetypal WC1 genes and WC1-11 with primer placement indicated. The WC1 common forward primer (WC1atg-for) for complete coding sequences was designed based on the conserved region in the signal sequences, while the reverse primers (WC1group1,2-rev and WC1group3-rev) were based on the end of the 3 ′ coding sequences. Abbreviations are as follows: ID, inter-domain sequence; TM, transmembrane region; ICD, intracytoplasmic domain. (B) cDNA evidence for WC1 genes. Primer pairs WC1atg-for/WC1group1,2-rev (designed for WC1group1,2) and primer set WC1atg-for/WC1group3-rev (designed for WC1group3) were used to amplify all the complete coding sequences of WC1 transcripts as described in the previous study [ 3 ]. (C) Confirmation of complete coding sequences for WC1-nd1 and WC1-nd2 . Four different templates used in PCR for all thirteen WC1 domain 1 specific primer pairs are indicated in the left part of each gel. (D) Agarose gel electrophoresis evidence for complete coding sequences of WC1-nd1 and WC1-nd2. Complete coding sequences of WC1-nd1 and WC1-nd2 amplified by primer pairs of specific forward primers and common reverse primers (WC1group1,2-rev). The cDNA isolated from sorted WC1.1 + γδ T cells was used as a template. Gel electrophoresis of the PCR products was performed on 1% agarose gel. For each primer set, the identity of the amplified products was confirmed by DNA sequencing analysis.
    Figure Legend Snippet: Amplification of full-length transcripts for WC1s. (A) Primers designed for complete coding sequences of all thirteen WC1 genes. Schematic representations of the molecular forms of archetypal WC1 genes and WC1-11 with primer placement indicated. The WC1 common forward primer (WC1atg-for) for complete coding sequences was designed based on the conserved region in the signal sequences, while the reverse primers (WC1group1,2-rev and WC1group3-rev) were based on the end of the 3 ′ coding sequences. Abbreviations are as follows: ID, inter-domain sequence; TM, transmembrane region; ICD, intracytoplasmic domain. (B) cDNA evidence for WC1 genes. Primer pairs WC1atg-for/WC1group1,2-rev (designed for WC1group1,2) and primer set WC1atg-for/WC1group3-rev (designed for WC1group3) were used to amplify all the complete coding sequences of WC1 transcripts as described in the previous study [ 3 ]. (C) Confirmation of complete coding sequences for WC1-nd1 and WC1-nd2 . Four different templates used in PCR for all thirteen WC1 domain 1 specific primer pairs are indicated in the left part of each gel. (D) Agarose gel electrophoresis evidence for complete coding sequences of WC1-nd1 and WC1-nd2. Complete coding sequences of WC1-nd1 and WC1-nd2 amplified by primer pairs of specific forward primers and common reverse primers (WC1group1,2-rev). The cDNA isolated from sorted WC1.1 + γδ T cells was used as a template. Gel electrophoresis of the PCR products was performed on 1% agarose gel. For each primer set, the identity of the amplified products was confirmed by DNA sequencing analysis.

    Techniques Used: Amplification, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Nucleic Acid Electrophoresis, DNA Sequencing

    Phylogenetic tree of WC1 domain 1 sequences. The evolutionary history of 90 taxa was inferred using Bayesian analysis in MrBayes3.2 [ 33 ]. Annotated sequences were designed with a prefix of “A”. Sequences obtained from individual animal of three breeds (Hereford, Belted Galloway, and Holstein) were designed with prefixes of “D”, “Y”, and “B”, respectively. Genomic DNA and transcripts sequences were designed with prefixes of “G and cDNA”, respectively. Markov chain Monte Carlo analysis was performed for 830,000 cycles, using 2 runs of 4 chains each, a temperature setting of 0.2, and an amino acid mixed model to approximate the posterior probabilities of trees, shown at branch nodes. The average standard deviation of split frequencies was 0.01, which was diagnostic of convergence at
    Figure Legend Snippet: Phylogenetic tree of WC1 domain 1 sequences. The evolutionary history of 90 taxa was inferred using Bayesian analysis in MrBayes3.2 [ 33 ]. Annotated sequences were designed with a prefix of “A”. Sequences obtained from individual animal of three breeds (Hereford, Belted Galloway, and Holstein) were designed with prefixes of “D”, “Y”, and “B”, respectively. Genomic DNA and transcripts sequences were designed with prefixes of “G and cDNA”, respectively. Markov chain Monte Carlo analysis was performed for 830,000 cycles, using 2 runs of 4 chains each, a temperature setting of 0.2, and an amino acid mixed model to approximate the posterior probabilities of trees, shown at branch nodes. The average standard deviation of split frequencies was 0.01, which was diagnostic of convergence at

    Techniques Used: Standard Deviation, Diagnostic Assay

    12) Product Images from "Sensitivity to TOP2 Targeting Chemotherapeutics Is Regulated by Oct1 and FILIP1L"

    Article Title: Sensitivity to TOP2 Targeting Chemotherapeutics Is Regulated by Oct1 and FILIP1L

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042921

    The transcription factor OCT1 mediates doxorubicin induced FILIP1L expression and apoptosis. OCT1, also called POU2F1 (POU domain, class 2, transcription factor 1), is a transcription factor that plays a role in the DNA damage response. We identified potential OCT1 binding sites in the FILIP1L promoter and tested if OCT1 is involved in Doxorubicin induced FILIP1L expression and apoptosis. (A) We targeted Oct1 for shRNA degradation in U2OS cells and used qPCR to verify 60% knockdown of target mRNA. Control and shOct1 cells were treated with 200 ng/ml doxorubicin and mRNA harvested 24 hours later for qPCR analysis. We determined that Oct1 mRNA levels are not affected by doxorubicin treatment. However, FILIP1L induction by doxorubicin is markedly reduced (∼65%) in shOct1 cells compared to control. (B) Control and shOct1 cells were treated with 400 ng/ml doxorubicin and measured for apoptosis at 24 hours. We found that Oct1 knockdown cells displayed 50% reduced doxorubicin induced apoptosis compared to control cells.
    Figure Legend Snippet: The transcription factor OCT1 mediates doxorubicin induced FILIP1L expression and apoptosis. OCT1, also called POU2F1 (POU domain, class 2, transcription factor 1), is a transcription factor that plays a role in the DNA damage response. We identified potential OCT1 binding sites in the FILIP1L promoter and tested if OCT1 is involved in Doxorubicin induced FILIP1L expression and apoptosis. (A) We targeted Oct1 for shRNA degradation in U2OS cells and used qPCR to verify 60% knockdown of target mRNA. Control and shOct1 cells were treated with 200 ng/ml doxorubicin and mRNA harvested 24 hours later for qPCR analysis. We determined that Oct1 mRNA levels are not affected by doxorubicin treatment. However, FILIP1L induction by doxorubicin is markedly reduced (∼65%) in shOct1 cells compared to control. (B) Control and shOct1 cells were treated with 400 ng/ml doxorubicin and measured for apoptosis at 24 hours. We found that Oct1 knockdown cells displayed 50% reduced doxorubicin induced apoptosis compared to control cells.

    Techniques Used: Expressing, Binding Assay, shRNA, Real-time Polymerase Chain Reaction

    A functional shRNA screen for regulators of doxorubicin induced apoptosis. (A) Outline of the doxorubicin induced apoptosis bypass screen using U2OS cells. Pools of shRNA were transfected into retroviral packaging cell lines, and retrovirus transduced into U2OS cells followed by puromycin selection. Transduced U2OS cells were treated with 225 ng/ml Doxorubicin for 5 days, which led to apoptotic death of approximately 99.8% of the library infected cells. We harvested cells that survived treatment, isolated genomic DNA, PCR amplified the region containing shRNA sequences, shotgun cloned and sequenced. A total of approximately 1500 inserts were sequenced. (B) Twelve genes identified by this screen are listed. Full gene names and the number of times identified are also listed.
    Figure Legend Snippet: A functional shRNA screen for regulators of doxorubicin induced apoptosis. (A) Outline of the doxorubicin induced apoptosis bypass screen using U2OS cells. Pools of shRNA were transfected into retroviral packaging cell lines, and retrovirus transduced into U2OS cells followed by puromycin selection. Transduced U2OS cells were treated with 225 ng/ml Doxorubicin for 5 days, which led to apoptotic death of approximately 99.8% of the library infected cells. We harvested cells that survived treatment, isolated genomic DNA, PCR amplified the region containing shRNA sequences, shotgun cloned and sequenced. A total of approximately 1500 inserts were sequenced. (B) Twelve genes identified by this screen are listed. Full gene names and the number of times identified are also listed.

    Techniques Used: Functional Assay, shRNA, Transfection, Selection, Infection, Isolation, Polymerase Chain Reaction, Amplification, Clone Assay

    Doxorubicin treatment induces FILIP1L expression. (A) U2OS cells were treated with 200 ng/ml doxorubicin and mRNA isolated 24 hours later for qPCR analysis. The twelve genes identified in the shRNA screen were tested for induction by doxorubicin. Expression of most genes was unaffected by doxorubicin treatment. However, two genes, expression of FILIP1L and HORMAD2 were significantly induced by doxorubicin treatment, particularly FILIP1L which showed > 200-fold induction. (B) FILIP1L induction by doxorubicin impaired following ATM/ATR inhibition in U2OS. Doxorubicin treatment induces DNA damage that activates the ATM and ATR kinases. Caffeine (4 mM) was used to inhibit ATM and ATR. FILIP1L induction by doxorubicin is reduced by over 90% by treatment with caffeine. SAOS-2 cells, which unlike U2OS do not contain wild-type p 53, fail to induce FILIP1L following doxorubicin treatment.
    Figure Legend Snippet: Doxorubicin treatment induces FILIP1L expression. (A) U2OS cells were treated with 200 ng/ml doxorubicin and mRNA isolated 24 hours later for qPCR analysis. The twelve genes identified in the shRNA screen were tested for induction by doxorubicin. Expression of most genes was unaffected by doxorubicin treatment. However, two genes, expression of FILIP1L and HORMAD2 were significantly induced by doxorubicin treatment, particularly FILIP1L which showed > 200-fold induction. (B) FILIP1L induction by doxorubicin impaired following ATM/ATR inhibition in U2OS. Doxorubicin treatment induces DNA damage that activates the ATM and ATR kinases. Caffeine (4 mM) was used to inhibit ATM and ATR. FILIP1L induction by doxorubicin is reduced by over 90% by treatment with caffeine. SAOS-2 cells, which unlike U2OS do not contain wild-type p 53, fail to induce FILIP1L following doxorubicin treatment.

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction, shRNA, Inhibition

    FILIP1L expression induces cell death. Ectopic expression of one of the identified genes, FILIP1L, caused significant induction of apoptosis on its own. U2OS and SAOS-2 cells were transfected with vector control (designated as “–” in the FILIP1L legend) or V5/His tagged FILIP1L expression plasmid. Cells were additionally treated with control or 200 ng/ml doxorubicin. Cells were harvested 24 hours after transfection and apoptotic cells were quantitated by measuring sub-G1 DNA content by propidium iodide staining. Apoptosis caused by FILIP1L expression in either cell type was not further augmented by treatment with doxorubicin.
    Figure Legend Snippet: FILIP1L expression induces cell death. Ectopic expression of one of the identified genes, FILIP1L, caused significant induction of apoptosis on its own. U2OS and SAOS-2 cells were transfected with vector control (designated as “–” in the FILIP1L legend) or V5/His tagged FILIP1L expression plasmid. Cells were additionally treated with control or 200 ng/ml doxorubicin. Cells were harvested 24 hours after transfection and apoptotic cells were quantitated by measuring sub-G1 DNA content by propidium iodide staining. Apoptosis caused by FILIP1L expression in either cell type was not further augmented by treatment with doxorubicin.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Staining

    13) Product Images from "The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions"

    Article Title: The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006875

    TcRad51 immunolocalization in T . cruzi . A) WT, TcRAD51 +/- and TcRAD51 ox cells are shown before irradiation (non-irradiated) and 24 h after exposure to 500 Gy of gamma irradiation. DNA is shown stained with DAPI and TcRad51 was detected using anti-TcRad51 antibody raised in mouse (diluted 1:2,000) and visualized with Alexa 555 conjugated goat-derived anti-mouse IgG secondary (diluted 1:5,000); black bar: 5 μm. Cells are also shown in phase contrast but at a lower magnification (white bar: 2 μm). A graphical representation of fluorescence intensities is shown alongside the images. Vertical bars indicate standard deviation, asterisks represent statistically significant differences (*** p
    Figure Legend Snippet: TcRad51 immunolocalization in T . cruzi . A) WT, TcRAD51 +/- and TcRAD51 ox cells are shown before irradiation (non-irradiated) and 24 h after exposure to 500 Gy of gamma irradiation. DNA is shown stained with DAPI and TcRad51 was detected using anti-TcRad51 antibody raised in mouse (diluted 1:2,000) and visualized with Alexa 555 conjugated goat-derived anti-mouse IgG secondary (diluted 1:5,000); black bar: 5 μm. Cells are also shown in phase contrast but at a lower magnification (white bar: 2 μm). A graphical representation of fluorescence intensities is shown alongside the images. Vertical bars indicate standard deviation, asterisks represent statistically significant differences (*** p

    Techniques Used: Irradiation, Staining, Derivative Assay, Fluorescence, Standard Deviation

    T . cruzi response to treatment with DNA cross-linking agents. Sensitivity of WT, TcRAD51 ox and TcRAD51 -/+ cells to: (A) 0, 500 J/m 2 or 1.000 J/m 2 of UV-light, or (B) 25 μM, 50 μM or 75 μM cisplatin. Parasites were counted 48 h after treatment. Numbers are represented as a percentage of untreated cells. Values represent the mean of triplicates. Error bars indicate standard deviations.
    Figure Legend Snippet: T . cruzi response to treatment with DNA cross-linking agents. Sensitivity of WT, TcRAD51 ox and TcRAD51 -/+ cells to: (A) 0, 500 J/m 2 or 1.000 J/m 2 of UV-light, or (B) 25 μM, 50 μM or 75 μM cisplatin. Parasites were counted 48 h after treatment. Numbers are represented as a percentage of untreated cells. Values represent the mean of triplicates. Error bars indicate standard deviations.

    Techniques Used:

    Growth of T . cruzi following DNA damage treatment. A) The sensitivity of WT and TcRAD51 -/+ cells to gamma radiation was determined. Parasites were exposed to 0 Gy or 500 Gy of gamma irradiation, the number of parasites was determined every two days for 28 d. Values represent the mean of triplicates and error bars indicate standard deviations. B) Chromosomal profile from different T . cruzi (WT, TcRAD51 ox, and TcRAD51 +/- ) samples after treatment with gamma radiation (500 Gy). Chromosomal bands from different T . cruzi samples were separated by PFGE and stained using ethidium bromide. Whole chromosomes from Saccharomyces cerevisiae were used as molecular weight markers. (C) T . cruzi growth curve after treatment with 20 mM HU. Arrow indicates the point when the drug was removed. (D) T . cruzi growth curve after treatment with 1.5 mM MMS. The number of cells was determined every 24 h by vital staining. Curves shown are an example of three independent experiments that were performed in triplicate. Error bars represent standard deviations. (E) Detection of H2A levels in epimastigotes treated with 20 mM HU. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot using anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%). (F) Detection of H2A levels in epimastigotes treated with 1.5 mM MMS. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot with anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%).
    Figure Legend Snippet: Growth of T . cruzi following DNA damage treatment. A) The sensitivity of WT and TcRAD51 -/+ cells to gamma radiation was determined. Parasites were exposed to 0 Gy or 500 Gy of gamma irradiation, the number of parasites was determined every two days for 28 d. Values represent the mean of triplicates and error bars indicate standard deviations. B) Chromosomal profile from different T . cruzi (WT, TcRAD51 ox, and TcRAD51 +/- ) samples after treatment with gamma radiation (500 Gy). Chromosomal bands from different T . cruzi samples were separated by PFGE and stained using ethidium bromide. Whole chromosomes from Saccharomyces cerevisiae were used as molecular weight markers. (C) T . cruzi growth curve after treatment with 20 mM HU. Arrow indicates the point when the drug was removed. (D) T . cruzi growth curve after treatment with 1.5 mM MMS. The number of cells was determined every 24 h by vital staining. Curves shown are an example of three independent experiments that were performed in triplicate. Error bars represent standard deviations. (E) Detection of H2A levels in epimastigotes treated with 20 mM HU. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot using anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%). (F) Detection of H2A levels in epimastigotes treated with 1.5 mM MMS. Cell lysates were separated by SDS-PAGE, and proteins were detected by Western blot with anti-H2A (1:3,000) antiserum and peroxidase-conjugated anti-IgG secondary (1:10,000 or 1:12,000). Loading control is depicted in the bottom panel (Ponceau 0.2%).

    Techniques Used: Irradiation, Staining, Molecular Weight, SDS Page, Western Blot

    14) Product Images from "Reactivation of Chromosomally Integrated Human Herpesvirus-6 by Telomeric Circle Formation"

    Article Title: Reactivation of Chromosomally Integrated Human Herpesvirus-6 by Telomeric Circle Formation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004033

    TRF2ΔB overexpression induces formation of circular HHV-6 genome in ciHHV-6 cells. ( A ) TRF2 and TRF2ΔB was over-expressed in an EBV immortalized cell line (PL-LCL) derived from blood cells of a ciHHV-6 person and in freshly isolated PBMCs of another ciHHV-6 person using lentiviral vectors. Protein expression was checked after 3 days of lentivirus infection by Western blot analysis. GAPDH served as loading control. *, Uncharacterized protein. ( B ) Telomere restriction fragment assay was performed in the same cells after 5 days of lentiviral infection using telomere specific probe. ( C ) Formation of circular extra-chromosomal HHV-6 DNA was detected in the same cells by inverse PCR. Data represents the PCR amplifications from PL-LCL cells. As a control PL-LCL cells were infected with C. trachomatis or heat inactivated Ctr (hCtr). Amplified PCR product, which was validated by sequencing, is marked with a black arrowhead.
    Figure Legend Snippet: TRF2ΔB overexpression induces formation of circular HHV-6 genome in ciHHV-6 cells. ( A ) TRF2 and TRF2ΔB was over-expressed in an EBV immortalized cell line (PL-LCL) derived from blood cells of a ciHHV-6 person and in freshly isolated PBMCs of another ciHHV-6 person using lentiviral vectors. Protein expression was checked after 3 days of lentivirus infection by Western blot analysis. GAPDH served as loading control. *, Uncharacterized protein. ( B ) Telomere restriction fragment assay was performed in the same cells after 5 days of lentiviral infection using telomere specific probe. ( C ) Formation of circular extra-chromosomal HHV-6 DNA was detected in the same cells by inverse PCR. Data represents the PCR amplifications from PL-LCL cells. As a control PL-LCL cells were infected with C. trachomatis or heat inactivated Ctr (hCtr). Amplified PCR product, which was validated by sequencing, is marked with a black arrowhead.

    Techniques Used: Over Expression, Derivative Assay, Isolation, Expressing, Infection, Western Blot, Inverse PCR, Polymerase Chain Reaction, Amplification, Sequencing

    PCR amplification of circular HHV-6 genomes having a single direct repeat (DR). ( A ) Location of primers and southern hybridization probes to detect each possible combination of circular viral DNA (see table S1 ). Different positions marked are based on the HHV-6A U1102 genome (X83413.1). ( B ) Total DNA was used for PCR using a primer pair facing against each other but located outside the viral DR (see table S1 ). PCR products were used for Southern hybridization using HHV-6 probe 2. The membrane was re-hybridized with HHV-6 probe 4 and later on with telomere (Tel-G) probe. P1–P5 represents 5 different DNA samples from ciHHV-6 individuals. P4+Ctr represents PBMCs from P4 after 2 weeks of Chlamydia infection. The position of molecular weight markers run is shown on the left.
    Figure Legend Snippet: PCR amplification of circular HHV-6 genomes having a single direct repeat (DR). ( A ) Location of primers and southern hybridization probes to detect each possible combination of circular viral DNA (see table S1 ). Different positions marked are based on the HHV-6A U1102 genome (X83413.1). ( B ) Total DNA was used for PCR using a primer pair facing against each other but located outside the viral DR (see table S1 ). PCR products were used for Southern hybridization using HHV-6 probe 2. The membrane was re-hybridized with HHV-6 probe 4 and later on with telomere (Tel-G) probe. P1–P5 represents 5 different DNA samples from ciHHV-6 individuals. P4+Ctr represents PBMCs from P4 after 2 weeks of Chlamydia infection. The position of molecular weight markers run is shown on the left.

    Techniques Used: Polymerase Chain Reaction, Amplification, Hybridization, Infection, Molecular Weight

    Detection of differentially processed ciHHV-6 ends. ( A ) Diagram of possible processing of the ciHHV-6 DR L -T2 end after viral integration. Approximate location of MboI digestion sites within and around viral DR are marked with dotted lines. The location of probes used to detect specific regions of viral DNA and their expected sizes are indicated. Different positions marked are based on HHV-6A U1102 genome (X83413.1). ( B ) Total genomic DNA from HSB-2 cells, with or without HHV-6A infection, were digested with MboI and processed for Southern hybridization. The membrane was stripped and re-probed sequentially with HHV-6 probe 2, Tel-G probe, HHV-6 probe 3 and finally with HHV-6 probe 1 (See table S1 ). ( C ) Similar experiment was performed in KBM-7 cells using HHV-6A probe 5, 6 and Tel-G probe. Chromosomal ends having DR L -T2 overhang is marked with red arrowhead. Short t-circles, which can be detected using Tel-G probe and HHV-6A probe 5 but not with HHV-6A probe 6 are marked with blue arrowhead. The positions of molecular weight markers are shown on the left. ( D ) Inverse PCR using forward and reverse primer directed out of the DR L (see Figure S4B for orientation). Equal amount of the PCR products were subjected to Southern hybridization using probes against telomeric repeats (Tel-G probe). The membrane was rehybridized with a second probe against viral DR (HHV-6 probe 1). P1–P5 represents 5 different DNA samples from ciHHV-6 individuals. P4+Ctr represents PBMCs from P4 after 2 weeks of Chlamydia infection. The positions of molecular weight markers are shown on the left. The bands, which hybridize with both the probes, represent the extra-chromosomal short t-circles (blue arrow head). The band marked with red arrowhead represents short t-circles with extremely short telomeric repeats, which do not hybridize with telomeric probe.
    Figure Legend Snippet: Detection of differentially processed ciHHV-6 ends. ( A ) Diagram of possible processing of the ciHHV-6 DR L -T2 end after viral integration. Approximate location of MboI digestion sites within and around viral DR are marked with dotted lines. The location of probes used to detect specific regions of viral DNA and their expected sizes are indicated. Different positions marked are based on HHV-6A U1102 genome (X83413.1). ( B ) Total genomic DNA from HSB-2 cells, with or without HHV-6A infection, were digested with MboI and processed for Southern hybridization. The membrane was stripped and re-probed sequentially with HHV-6 probe 2, Tel-G probe, HHV-6 probe 3 and finally with HHV-6 probe 1 (See table S1 ). ( C ) Similar experiment was performed in KBM-7 cells using HHV-6A probe 5, 6 and Tel-G probe. Chromosomal ends having DR L -T2 overhang is marked with red arrowhead. Short t-circles, which can be detected using Tel-G probe and HHV-6A probe 5 but not with HHV-6A probe 6 are marked with blue arrowhead. The positions of molecular weight markers are shown on the left. ( D ) Inverse PCR using forward and reverse primer directed out of the DR L (see Figure S4B for orientation). Equal amount of the PCR products were subjected to Southern hybridization using probes against telomeric repeats (Tel-G probe). The membrane was rehybridized with a second probe against viral DR (HHV-6 probe 1). P1–P5 represents 5 different DNA samples from ciHHV-6 individuals. P4+Ctr represents PBMCs from P4 after 2 weeks of Chlamydia infection. The positions of molecular weight markers are shown on the left. The bands, which hybridize with both the probes, represent the extra-chromosomal short t-circles (blue arrow head). The band marked with red arrowhead represents short t-circles with extremely short telomeric repeats, which do not hybridize with telomeric probe.

    Techniques Used: Infection, Hybridization, Molecular Weight, Inverse PCR, Polymerase Chain Reaction

    15) Product Images from "Isolation and Characterization of Polymorphic DNA from Entamoeba histolytica"

    Article Title: Isolation and Characterization of Polymorphic DNA from Entamoeba histolytica

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.39.3.897-905.2001

    Polymorphic DNA analysis of Entamoeba histolytica isolates. (A) Locus 9-4. Amplification products were generated using primers R9 and R4 at an annealing temperature of 55°C. (B) Half-locus 9-11. Amplification products were generated using primers R9 and R11 at an annealing temperature of 50°C. (C) Half-locus 10-4. Amplification products were generated using primers R10 and R4 at an annealing temperature of 50°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).
    Figure Legend Snippet: Polymorphic DNA analysis of Entamoeba histolytica isolates. (A) Locus 9-4. Amplification products were generated using primers R9 and R4 at an annealing temperature of 55°C. (B) Half-locus 9-11. Amplification products were generated using primers R9 and R11 at an annealing temperature of 50°C. (C) Half-locus 10-4. Amplification products were generated using primers R10 and R4 at an annealing temperature of 50°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).

    Techniques Used: Amplification, Generated

    Polymorphic DNA analysis of E. histolytica isolates. (A) Locus 16-17. Amplification products were generated using primers R16 and R17 at an annealing temperature of 55°C. (B) Half-locus 16-19. Amplification products were generated using primers R16 and R19 at an annealing temperature of 54°C. (C) Half-locus 18-17. Amplification products were generated using primers R18 and R17 at an annealing temperature of 54°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).
    Figure Legend Snippet: Polymorphic DNA analysis of E. histolytica isolates. (A) Locus 16-17. Amplification products were generated using primers R16 and R17 at an annealing temperature of 55°C. (B) Half-locus 16-19. Amplification products were generated using primers R16 and R19 at an annealing temperature of 54°C. (C) Half-locus 18-17. Amplification products were generated using primers R18 and R17 at an annealing temperature of 54°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).

    Techniques Used: Amplification, Generated

    Polymorphic DNA analysis of Entamoeba histolytica isolates. (A) Locus 1-2. Amplification products were generated using primers R1 and R2 at an annealing temperature of 53°C. (B) Locus 5-6. Amplification products were generated using primers R5A and R6A at an annealing temperature of 56°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).
    Figure Legend Snippet: Polymorphic DNA analysis of Entamoeba histolytica isolates. (A) Locus 1-2. Amplification products were generated using primers R1 and R2 at an annealing temperature of 53°C. (B) Locus 5-6. Amplification products were generated using primers R5A and R6A at an annealing temperature of 56°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).

    Techniques Used: Amplification, Generated

    Polymorphic DNA analysis of E. histolytica isolates. (A) Locus 3-4. Amplification products were generated using primers R3 and R4 at an annealing temperature of 55°C. (B) Half-locus 3-8. Amplification products were generated using primers R3 and R8 at an annealing temperature of 50°C. (C) Half-locus 7-4. Amplification products were generated using primers R7 and R4 at an annealing temperature of 50°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).
    Figure Legend Snippet: Polymorphic DNA analysis of E. histolytica isolates. (A) Locus 3-4. Amplification products were generated using primers R3 and R4 at an annealing temperature of 55°C. (B) Half-locus 3-8. Amplification products were generated using primers R3 and R8 at an annealing temperature of 50°C. (C) Half-locus 7-4. Amplification products were generated using primers R7 and R4 at an annealing temperature of 50°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).

    Techniques Used: Amplification, Generated

    16) Product Images from "Genetic Authentication of Gardenia jasminoides Ellis var. grandiflora Nakai by Improved RAPD-Derived DNA Markers"

    Article Title: Genetic Authentication of Gardenia jasminoides Ellis var. grandiflora Nakai by Improved RAPD-Derived DNA Markers

    Journal: Molecules

    doi: 10.3390/molecules201119687

    Genetic authentication of Gardenia jasminoides based on ZZH11 ( A ); ZZH31 ( B ); ZZH41 ( C ) and ZZH51 ( D ) SCAR markers. Lanes 1–6 are the DNA samples of G. jasminoides Ellis var. grandiflora Nakai from Jiangxi, Fujian, Guizhou, Sichuan, Hunan and Zhejiang respectively ( Table 1 ). Lane 7 is a sample of G. jasminoides from Luzhou in Sichuan (No. 7 in Table 1 ). Lane M indicates the DNA molecular weight marker DL600 with the fragment size (bp).
    Figure Legend Snippet: Genetic authentication of Gardenia jasminoides based on ZZH11 ( A ); ZZH31 ( B ); ZZH41 ( C ) and ZZH51 ( D ) SCAR markers. Lanes 1–6 are the DNA samples of G. jasminoides Ellis var. grandiflora Nakai from Jiangxi, Fujian, Guizhou, Sichuan, Hunan and Zhejiang respectively ( Table 1 ). Lane 7 is a sample of G. jasminoides from Luzhou in Sichuan (No. 7 in Table 1 ). Lane M indicates the DNA molecular weight marker DL600 with the fragment size (bp).

    Techniques Used: Molecular Weight, Marker

    Genetic authentication of more Gardenia jasminoides sample based on ZZH11 ( A ); ZZH31 ( B ); ZZH41 ( C ) and ZZH51 ( D ) SCAR markers. Lanes 6, 8, 9, 10 and 11 are the DNA samples of G. jasminoides Ellis var. grandiflora Nakai from Zhejiang, Guangdong, Fujian, Hunan and Anhui respectively ( Table 1 , Nos. 6, 8, 9, 10 and 11). Lane 7, 12, 13, 14 and 15 are samples of G. jasminoides from Luzhou in Sichuan, Jiangsu, Jiangxi, Hunan and Shanxi respectively ( Table 1 , Nos., 7, 12, 13, 14 and 15). Lane M indicates the DNA molecular weight marker DL600 with the fragment size (bp).
    Figure Legend Snippet: Genetic authentication of more Gardenia jasminoides sample based on ZZH11 ( A ); ZZH31 ( B ); ZZH41 ( C ) and ZZH51 ( D ) SCAR markers. Lanes 6, 8, 9, 10 and 11 are the DNA samples of G. jasminoides Ellis var. grandiflora Nakai from Zhejiang, Guangdong, Fujian, Hunan and Anhui respectively ( Table 1 , Nos. 6, 8, 9, 10 and 11). Lane 7, 12, 13, 14 and 15 are samples of G. jasminoides from Luzhou in Sichuan, Jiangsu, Jiangxi, Hunan and Shanxi respectively ( Table 1 , Nos., 7, 12, 13, 14 and 15). Lane M indicates the DNA molecular weight marker DL600 with the fragment size (bp).

    Techniques Used: Molecular Weight, Marker

    Development of stable RAPD-sequence-characterized amplified region (SCAR) markers for ZZH11, ZZH31, ZZH41 and ZZH51. ( A ) A SCAR marker ZZH11; ( B ) A SCAR marker ZZH31; ( C ) A SCAR marker ZZH41; ( D ) A SCAR marker ZZH51. Lanes 1~6 indicate the different samples of G. jasminoides Ellis var. grandiflora Nakai listed in Table 1 . Lanes 7 and 8 are two samples of Lonicera japonica from Hunan and Hubei; Lane 9 is one sample of Penthorum chinense ; Lane 10 is one sample of Ganoderma lucidum ; Lanes 11 and 12 are two samples of Litchi chinesis from Guangdong and Sichuan; Lanes 13 and 14 are two samples of Dimocarpus Longan from Fujian and Sichuan; Lane 15 is one sample of Dimocarpus confinis from Guangxi; Lanes 16 and 17 are two samples of Ginkgo Biloba from Sichuan and Hunan; Lanes 18 and 19 are two samples of Angelica sinensis from Sichuan and Gansu; Lane 20 is one sample of Gastrodia elata ; Lanes 21 and 22 are two samples of Canarium album Guangdong and Sichuan; Lane 23 is negative control without DNA. The blue arrows indicate expected PCR products in size. Lane M indicates the DNA molecular weight marker DL2000.
    Figure Legend Snippet: Development of stable RAPD-sequence-characterized amplified region (SCAR) markers for ZZH11, ZZH31, ZZH41 and ZZH51. ( A ) A SCAR marker ZZH11; ( B ) A SCAR marker ZZH31; ( C ) A SCAR marker ZZH41; ( D ) A SCAR marker ZZH51. Lanes 1~6 indicate the different samples of G. jasminoides Ellis var. grandiflora Nakai listed in Table 1 . Lanes 7 and 8 are two samples of Lonicera japonica from Hunan and Hubei; Lane 9 is one sample of Penthorum chinense ; Lane 10 is one sample of Ganoderma lucidum ; Lanes 11 and 12 are two samples of Litchi chinesis from Guangdong and Sichuan; Lanes 13 and 14 are two samples of Dimocarpus Longan from Fujian and Sichuan; Lane 15 is one sample of Dimocarpus confinis from Guangxi; Lanes 16 and 17 are two samples of Ginkgo Biloba from Sichuan and Hunan; Lanes 18 and 19 are two samples of Angelica sinensis from Sichuan and Gansu; Lane 20 is one sample of Gastrodia elata ; Lanes 21 and 22 are two samples of Canarium album Guangdong and Sichuan; Lane 23 is negative control without DNA. The blue arrows indicate expected PCR products in size. Lane M indicates the DNA molecular weight marker DL2000.

    Techniques Used: Sequencing, Amplification, Marker, Negative Control, Polymerase Chain Reaction, Molecular Weight

    17) Product Images from "Characterization and diagnostic application of genomic NPM-ALK fusion sequences in anaplastic large-cell lymphoma"

    Article Title: Characterization and diagnostic application of genomic NPM-ALK fusion sequences in anaplastic large-cell lymphoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25489

    NPM-ALK quantification after crizotinib treatment in vitro Parallel quantification of NPM-ALK fusion transcript (A) and fusion gene (B) copies and cell survival analyses (D) after treatment of ALK-positive ALCL cell lines (SR-786 or Karpas 299) mixed 1:100 with an ALK-negative lymphoma cell line (DG75) with crizotinib for 72 h. Transcripts per cell were calculated based on quantified RNA and DNA copies (C) . Experiments were repeated three or five times for SR-786 and Karpas 299, respectively. ( **** p
    Figure Legend Snippet: NPM-ALK quantification after crizotinib treatment in vitro Parallel quantification of NPM-ALK fusion transcript (A) and fusion gene (B) copies and cell survival analyses (D) after treatment of ALK-positive ALCL cell lines (SR-786 or Karpas 299) mixed 1:100 with an ALK-negative lymphoma cell line (DG75) with crizotinib for 72 h. Transcripts per cell were calculated based on quantified RNA and DNA copies (C) . Experiments were repeated three or five times for SR-786 and Karpas 299, respectively. ( **** p

    Techniques Used: In Vitro

    Comparison of quantitative NPM-ALK PCR results between cellular RNA, cellular DNA and cell free DNA Comparison of NPM-ALK copy numbers in blood or bone marrow samples from high risk ALCL-patients using the cellular RNA- and DNA-based and cell-free DNA-based methods. (A) Cell-based fusion transcripts (RNA) versus cellular fusion-sequence DNA-based (DNA), n=45. (B) Cell-based fusion transcripts (RNA) versus cell-free fusion-sequence DNA-based (ctDNA), n=37. (C) Cellular fusion-sequence DNA-based (DNA) versus cell-free fusion-sequence DNA-based (ctDNA), n=37.
    Figure Legend Snippet: Comparison of quantitative NPM-ALK PCR results between cellular RNA, cellular DNA and cell free DNA Comparison of NPM-ALK copy numbers in blood or bone marrow samples from high risk ALCL-patients using the cellular RNA- and DNA-based and cell-free DNA-based methods. (A) Cell-based fusion transcripts (RNA) versus cellular fusion-sequence DNA-based (DNA), n=45. (B) Cell-based fusion transcripts (RNA) versus cell-free fusion-sequence DNA-based (ctDNA), n=37. (C) Cellular fusion-sequence DNA-based (DNA) versus cell-free fusion-sequence DNA-based (ctDNA), n=37.

    Techniques Used: Polymerase Chain Reaction, Sequencing

    Quantification of NPM-ALK in ALCL patients NPM-ALK fusion RNA, DNA and ctDNA copies in 3 ALCL patients during their disease course. (AM… course of dexamethasone, methotrexate, ifosfamide, cytarabine and etoposide; BM… course of dexamethasone, methotrexate, cyclophosphamide and doxorubicine).
    Figure Legend Snippet: Quantification of NPM-ALK in ALCL patients NPM-ALK fusion RNA, DNA and ctDNA copies in 3 ALCL patients during their disease course. (AM… course of dexamethasone, methotrexate, ifosfamide, cytarabine and etoposide; BM… course of dexamethasone, methotrexate, cyclophosphamide and doxorubicine).

    Techniques Used:

    18) Product Images from "Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector"

    Article Title: Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1608013113

    DNA and RNA Extraction; RT-, qRT-, and q-PCR; and Southern/Northern Hybridization Blots.
    Figure Legend Snippet: DNA and RNA Extraction; RT-, qRT-, and q-PCR; and Southern/Northern Hybridization Blots.

    Techniques Used: RNA Extraction, Polymerase Chain Reaction, Northern Blot, Hybridization

    Detection of SsHADV-1 from L. ingenua ( A ) and second-passage Sf9 cells ( B and C ). ( A ) RT-PCR for detecting the expression of the SsHADV-1 Rep and CP gene in L. ingenua . Lane M, DNA marker; lanes 1–6, RNA samples of strain DT-8 (positive control);
    Figure Legend Snippet: Detection of SsHADV-1 from L. ingenua ( A ) and second-passage Sf9 cells ( B and C ). ( A ) RT-PCR for detecting the expression of the SsHADV-1 Rep and CP gene in L. ingenua . Lane M, DNA marker; lanes 1–6, RNA samples of strain DT-8 (positive control);

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Positive Control

    Replication of SsHADV-1 in S. frugiperda cells (Sf9). ( A ) PCR amplification of SsHADV-1 in virus-inoculated Sf9 cells. Viral DNA was amplified from virus-inoculated Sf9 cells (indicated by white arrow). Lane M, DNA marker; lanes 1–3, DNA from
    Figure Legend Snippet: Replication of SsHADV-1 in S. frugiperda cells (Sf9). ( A ) PCR amplification of SsHADV-1 in virus-inoculated Sf9 cells. Viral DNA was amplified from virus-inoculated Sf9 cells (indicated by white arrow). Lane M, DNA marker; lanes 1–3, DNA from

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker

    19) Product Images from "A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps"

    Article Title: A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064084

    Combining NGS library preparation with size selection on the AMCC chip. Columns were formed on the AMCC chip using carboxylated beads. (A) Electropherogram showing size selection on-chip, illustrating the recovery of DNA fragments in the range of 150-500 bp. The green trace represents the DNA ladder used. GEL image extracted from a high sensitivity chip run on the Bioanalyzer 2100. (B) Quantification of Illumina library DNA eluted from the AMCC chip using RT-qPCR. Coefficient of variation (CV) for this chip run was 0.05. Asterisks indicate sample modules where buffer was loaded instead of genomic DNA. (C) Coverage depths from sequencing runs of libraries prepared on the AMCC chip and using the conventional benchtop protocol recommended by the manufacturer.
    Figure Legend Snippet: Combining NGS library preparation with size selection on the AMCC chip. Columns were formed on the AMCC chip using carboxylated beads. (A) Electropherogram showing size selection on-chip, illustrating the recovery of DNA fragments in the range of 150-500 bp. The green trace represents the DNA ladder used. GEL image extracted from a high sensitivity chip run on the Bioanalyzer 2100. (B) Quantification of Illumina library DNA eluted from the AMCC chip using RT-qPCR. Coefficient of variation (CV) for this chip run was 0.05. Asterisks indicate sample modules where buffer was loaded instead of genomic DNA. (C) Coverage depths from sequencing runs of libraries prepared on the AMCC chip and using the conventional benchtop protocol recommended by the manufacturer.

    Techniques Used: Next-Generation Sequencing, Selection, Chromatin Immunoprecipitation, Quantitative RT-PCR, Sequencing

    Microfluidic device for automated NGS library preparation. (a) Automated multi-column device mounted on a plastic carrier that provides wells for loading samples and reagents and for pressurized operation of the device. The wells used to load reagents for NGS library preparation are labeled. Chromatography columns for the selective binding and release of DNA were formed either with ChargeSwitch beads or with carboxylated beads. Reagents that were used exclusively with the carboxylated beads are labeled in green. (b) Schematic of single reactor unit for reaction mixing and DNA purification. The regions denoted in the reaction circuit are as follows: Green, Sample; Orange, Buffer; Blue, Enzyme. Red solid rectangular boxes represent activated valves that partition the individual circuits. (c) Parallelization of 16 reactors on chip for preparation of up to 16 independent libraries. Layout of the entire device without the valve map showing reagent inlets and the design for multiplex library generation. The serpentine metering channel designed to ensure reliable column packing is highlighted in orange. (d) Schematics showing cross-sections of purification columns loaded with either 1) ChargeSwitch beads, which are held in place with a frit layer and a cap layer formed by larger beads, or 2) carboxylated beads.
    Figure Legend Snippet: Microfluidic device for automated NGS library preparation. (a) Automated multi-column device mounted on a plastic carrier that provides wells for loading samples and reagents and for pressurized operation of the device. The wells used to load reagents for NGS library preparation are labeled. Chromatography columns for the selective binding and release of DNA were formed either with ChargeSwitch beads or with carboxylated beads. Reagents that were used exclusively with the carboxylated beads are labeled in green. (b) Schematic of single reactor unit for reaction mixing and DNA purification. The regions denoted in the reaction circuit are as follows: Green, Sample; Orange, Buffer; Blue, Enzyme. Red solid rectangular boxes represent activated valves that partition the individual circuits. (c) Parallelization of 16 reactors on chip for preparation of up to 16 independent libraries. Layout of the entire device without the valve map showing reagent inlets and the design for multiplex library generation. The serpentine metering channel designed to ensure reliable column packing is highlighted in orange. (d) Schematics showing cross-sections of purification columns loaded with either 1) ChargeSwitch beads, which are held in place with a frit layer and a cap layer formed by larger beads, or 2) carboxylated beads.

    Techniques Used: Next-Generation Sequencing, Labeling, Chromatography, Binding Assay, DNA Purification, Chromatin Immunoprecipitation, Multiplex Assay, Purification

    Quantification of E. coli strain DH10B library DNA after size selection. (a) Illumina libraries; (b) Ion Torrent libraries. Asterisks indicate sample modules where buffer was loaded instead of genomic DNA. (c) Efficiency of library preparation reactions on the AMCC chip. The percentage of E. coli DNA fragments with Illumina sequencing adapters ligated onto both ends was estimated by RT-qPCR. The amount of E. coli genomic DNA present was determined by RT-qPCR with primer pairs recognizing six regions of the E. coli genome, and the amount of library DNA with adapters ligated onto both ends was determined by RT-qPCR with a primer pair recognizing the Illumina sequencing adapters. RT-qPCR data were converted to nanograms of DNA using standard curves (Materials and Methods). The estimated amounts of E. coli genomic DNA present in each library varied somewhat between the six locus-specific RT-qPCR reactions, so the bar graph indicates the mean values, and the error bars indicate the standard error of the mean.
    Figure Legend Snippet: Quantification of E. coli strain DH10B library DNA after size selection. (a) Illumina libraries; (b) Ion Torrent libraries. Asterisks indicate sample modules where buffer was loaded instead of genomic DNA. (c) Efficiency of library preparation reactions on the AMCC chip. The percentage of E. coli DNA fragments with Illumina sequencing adapters ligated onto both ends was estimated by RT-qPCR. The amount of E. coli genomic DNA present was determined by RT-qPCR with primer pairs recognizing six regions of the E. coli genome, and the amount of library DNA with adapters ligated onto both ends was determined by RT-qPCR with a primer pair recognizing the Illumina sequencing adapters. RT-qPCR data were converted to nanograms of DNA using standard curves (Materials and Methods). The estimated amounts of E. coli genomic DNA present in each library varied somewhat between the six locus-specific RT-qPCR reactions, so the bar graph indicates the mean values, and the error bars indicate the standard error of the mean.

    Techniques Used: Selection, Chromatin Immunoprecipitation, Sequencing, Quantitative RT-PCR

    20) Product Images from "Association of Activating KIR Copy Number Variation of NK Cells with Containment of SIV Replication in Rhesus Monkeys"

    Article Title: Association of Activating KIR Copy Number Variation of NK Cells with Containment of SIV Replication in Rhesus Monkeys

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002436

    Association of KIR3DH copy numbers and peak plasma SIV RNA levels in Indian-origin rhesus monkeys. KIR3DH copy numbers were determined by quantitative real-time PCR using genomic DNA from rhesus monkeys. Peak plasma SIV RNA levels were quantified on day 14 post-SIVmac251-infection. Scatter plots represent the relationship between SIV peak viral load and KIR3DH copy numbers in the entire cohort of rhesus monkeys ( P = 0.70) (A), in Mamu-A*01 + rhesus monkeys ( P = 0.24) (B) and in Mamu-A*01 – rhesus monkeys ( P = 0.08) (C). The Mamu-A*01 + and Mamu-A*01 – rhesus monkeys were subdivided into two groups: one group having KIR3DH copy numbers below the median (black) and the other group having KIR3DH copy numbers above the median (blue). These groups were further subdivided into monkeys expressing only TRIM5 alleles 1–5 or expressing at least one TRIM5 allele of the group 6–11. P values were determined using the Mann-Whitney U test (two-tailed).
    Figure Legend Snippet: Association of KIR3DH copy numbers and peak plasma SIV RNA levels in Indian-origin rhesus monkeys. KIR3DH copy numbers were determined by quantitative real-time PCR using genomic DNA from rhesus monkeys. Peak plasma SIV RNA levels were quantified on day 14 post-SIVmac251-infection. Scatter plots represent the relationship between SIV peak viral load and KIR3DH copy numbers in the entire cohort of rhesus monkeys ( P = 0.70) (A), in Mamu-A*01 + rhesus monkeys ( P = 0.24) (B) and in Mamu-A*01 – rhesus monkeys ( P = 0.08) (C). The Mamu-A*01 + and Mamu-A*01 – rhesus monkeys were subdivided into two groups: one group having KIR3DH copy numbers below the median (black) and the other group having KIR3DH copy numbers above the median (blue). These groups were further subdivided into monkeys expressing only TRIM5 alleles 1–5 or expressing at least one TRIM5 allele of the group 6–11. P values were determined using the Mann-Whitney U test (two-tailed).

    Techniques Used: Real-time Polymerase Chain Reaction, Infection, Expressing, MANN-WHITNEY, Two Tailed Test

    Distribution of KIR3DH copy numbers in Indian-origin rhesus monkeys. Copy numbers of KIR3DH genes were determined using quantitative real-time PCR on genomic DNA of 77 Indian-origin rhesus monkeys. (A) Distribution of KIR3DH copy numbers in the entire cohort of monkeys. (B) Boxplots of KIR3DH copy number distribution in rhesus monkeys divided into three cohorts: Mamu-A*01 – and Mamu-A*01 + rhesus monkeys, Mamu-B*17 – and Mamu-B*17 + rhesus monkeys and rhesus monkeys expressing only TRIM5 alleles 1–5 or expressing at least one TRIM5 allele from the group 6–11. (C) The median, mean and standard deviation (SD) of KIR3DH copy numbers are shown for various subgroups of rhesus monkeys. P values were calculated using the Mann-Whitney U test (two-tailed).
    Figure Legend Snippet: Distribution of KIR3DH copy numbers in Indian-origin rhesus monkeys. Copy numbers of KIR3DH genes were determined using quantitative real-time PCR on genomic DNA of 77 Indian-origin rhesus monkeys. (A) Distribution of KIR3DH copy numbers in the entire cohort of monkeys. (B) Boxplots of KIR3DH copy number distribution in rhesus monkeys divided into three cohorts: Mamu-A*01 – and Mamu-A*01 + rhesus monkeys, Mamu-B*17 – and Mamu-B*17 + rhesus monkeys and rhesus monkeys expressing only TRIM5 alleles 1–5 or expressing at least one TRIM5 allele from the group 6–11. (C) The median, mean and standard deviation (SD) of KIR3DH copy numbers are shown for various subgroups of rhesus monkeys. P values were calculated using the Mann-Whitney U test (two-tailed).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, MANN-WHITNEY, Two Tailed Test

    Intra-run reproducibility in KIR3DH copy number determination and validation of quantitative real-time PCR estimates of KIR3DH copy numbers by MLPA (multiplex ligation-dependent probe amplification). (A) KIR3DH copy numbers were determined using triplicates of each DNA sample from 77 rhesus monkeys in two separate experiments to validate intra-experiment reproducibility (R 2 = 0.87, β = 0.816). (B) Comparison of signal ratios of two MLPA experiments. Pairs of ratios (20 samples) cluster around groups corresponding to KIR3DH copy numbers of 2, 3, 4 and 5. (C) Relationship between KIR3DH copy numbers determined by qPCR and MLPA (R 2 = 0.77, β = 0.729). The 95% confidence interval is shown by dashed lines.
    Figure Legend Snippet: Intra-run reproducibility in KIR3DH copy number determination and validation of quantitative real-time PCR estimates of KIR3DH copy numbers by MLPA (multiplex ligation-dependent probe amplification). (A) KIR3DH copy numbers were determined using triplicates of each DNA sample from 77 rhesus monkeys in two separate experiments to validate intra-experiment reproducibility (R 2 = 0.87, β = 0.816). (B) Comparison of signal ratios of two MLPA experiments. Pairs of ratios (20 samples) cluster around groups corresponding to KIR3DH copy numbers of 2, 3, 4 and 5. (C) Relationship between KIR3DH copy numbers determined by qPCR and MLPA (R 2 = 0.77, β = 0.729). The 95% confidence interval is shown by dashed lines.

    Techniques Used: Real-time Polymerase Chain Reaction, Multiplex Ligation-dependent Probe Amplification, Multiplex Assay, Ligation, Amplification

    21) Product Images from "A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models"

    Article Title: A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158597

    GnRH-E1 RNA is localized in the GT1-7 neuron nucleus. (A) Schematic diagram of the conserved regulatory elements upstream of the mouse Gnrh1 TSS, which contains enhancers 1, 2, and 3 (E3, E2, E1, respectively), the promoter (P), and the Gnrh1 gene with four exons (white boxes). Coordinates above the regulatory elements indicate positions with respect to the Gnrh1 TSS. RT-PCR primers used in B-D are indicated by arrows, and expected PCR products are represented by a connecting line. Positions of PCR primers are aligned to the mouse conserved regulatory region diagrammed above. Nuclear and cytoplasmic extracts from GT1-7 neurons were analyzed for GnRH-E1 RNA (B), Gnrh1 pre-mRNA (C), Gnrh1 mRNA (D), and H2afz mRNA control (E) by RT-PCR. RT-PCR analysis was performed on random hexamer-primed cDNA, where cDNA synthesized with (+) and without (-) reverse transcriptase were analyzed in parallel. PCR loading controls are plasmid containing the -3568/-1128 bp segment upstream of the Gnrh1 TSS and no-template control (NTC). The sizes of the PCR amplicons were marked by a 100 bp DNA ladder or a 1 kbp DNA ladder where indicated, that were resolved on the agarose gel in parallel.
    Figure Legend Snippet: GnRH-E1 RNA is localized in the GT1-7 neuron nucleus. (A) Schematic diagram of the conserved regulatory elements upstream of the mouse Gnrh1 TSS, which contains enhancers 1, 2, and 3 (E3, E2, E1, respectively), the promoter (P), and the Gnrh1 gene with four exons (white boxes). Coordinates above the regulatory elements indicate positions with respect to the Gnrh1 TSS. RT-PCR primers used in B-D are indicated by arrows, and expected PCR products are represented by a connecting line. Positions of PCR primers are aligned to the mouse conserved regulatory region diagrammed above. Nuclear and cytoplasmic extracts from GT1-7 neurons were analyzed for GnRH-E1 RNA (B), Gnrh1 pre-mRNA (C), Gnrh1 mRNA (D), and H2afz mRNA control (E) by RT-PCR. RT-PCR analysis was performed on random hexamer-primed cDNA, where cDNA synthesized with (+) and without (-) reverse transcriptase were analyzed in parallel. PCR loading controls are plasmid containing the -3568/-1128 bp segment upstream of the Gnrh1 TSS and no-template control (NTC). The sizes of the PCR amplicons were marked by a 100 bp DNA ladder or a 1 kbp DNA ladder where indicated, that were resolved on the agarose gel in parallel.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Random Hexamer Labeling, Synthesized, Plasmid Preparation, Agarose Gel Electrophoresis

    Strand-specific cDNA and RT-PCR analysis of the mouse GnRH-E1 RNA variants. (A) Schematic diagram of the sense GnRH-E1 RNA variant, with a 3’ polyA site located downstream of GnRH-E1 as predicted by RACE. PCR primers in reverse direction at -1128 bp or -1271 bp (reverse arrows) were used for strand-specific cDNA synthesis to capture the sense GnRH-E1 RNA. PCR analysis was performed using -1271 bp or -1128 bp reverse primer paired with the forward primers at -3560 bp, -3606 bp, -3779 bp, and -3746 bp (forward arrows) from the mouse Gnrh1 TSS. Primer positions are aligned to the mouse conserved regulatory elements and coordinates diagrammed above. (B) Strand-specific cDNA synthesized using the -1271 bp reverse primer was subject to PCR analysis using the following primer pairs: -3560F/-1271R, -3606F/-1271R, -3779F/-1271R, -3746F/-1271R. Strand-specific cDNA synthesized using -1128 bp reverse primer was subject to PCR analysis using the primer pair -3560F/-1128R. (C) Schematic diagram of the mouse antisense GnRH-E1 RNA variant, with a 3’ polyA site predicted upstream of GnRH-E2 by RACE. PCR primers in the forward direction at -3560 bp or -3371 bp (forward arrows) was used for strand-specific cDNA synthesis to capture the antisense GnRH-E1 RNA variant. PCR analysis was performed using -3371 bp or -3560 bp forward primer paired with the reverse primers at -1443 bp, -1271 bp, -1128 bp from the Gnrh1 TSS. D) Strand-specific cDNA synthesized using the -3371 bp forward primer was subject to PCR analysis using the following PCR primer pairs -3371F/-1443R, -3371F/-1271R, and -3371F/-1128R. Strand-specific cDNA synthesized using the -3560 bp forward primer was subject to PCR analysis using the primer pair at -3560F/-1443R. All reverse transcription reactions were performed on total RNA samples with (+) and without (-) reverse transcriptase and were amplified by PCR in parallel with GT1-7 genomic DNA control and no-template water control (NTC). The size of PCR amplicons was marked by 1 kbp DNA ladder.
    Figure Legend Snippet: Strand-specific cDNA and RT-PCR analysis of the mouse GnRH-E1 RNA variants. (A) Schematic diagram of the sense GnRH-E1 RNA variant, with a 3’ polyA site located downstream of GnRH-E1 as predicted by RACE. PCR primers in reverse direction at -1128 bp or -1271 bp (reverse arrows) were used for strand-specific cDNA synthesis to capture the sense GnRH-E1 RNA. PCR analysis was performed using -1271 bp or -1128 bp reverse primer paired with the forward primers at -3560 bp, -3606 bp, -3779 bp, and -3746 bp (forward arrows) from the mouse Gnrh1 TSS. Primer positions are aligned to the mouse conserved regulatory elements and coordinates diagrammed above. (B) Strand-specific cDNA synthesized using the -1271 bp reverse primer was subject to PCR analysis using the following primer pairs: -3560F/-1271R, -3606F/-1271R, -3779F/-1271R, -3746F/-1271R. Strand-specific cDNA synthesized using -1128 bp reverse primer was subject to PCR analysis using the primer pair -3560F/-1128R. (C) Schematic diagram of the mouse antisense GnRH-E1 RNA variant, with a 3’ polyA site predicted upstream of GnRH-E2 by RACE. PCR primers in the forward direction at -3560 bp or -3371 bp (forward arrows) was used for strand-specific cDNA synthesis to capture the antisense GnRH-E1 RNA variant. PCR analysis was performed using -3371 bp or -3560 bp forward primer paired with the reverse primers at -1443 bp, -1271 bp, -1128 bp from the Gnrh1 TSS. D) Strand-specific cDNA synthesized using the -3371 bp forward primer was subject to PCR analysis using the following PCR primer pairs -3371F/-1443R, -3371F/-1271R, and -3371F/-1128R. Strand-specific cDNA synthesized using the -3560 bp forward primer was subject to PCR analysis using the primer pair at -3560F/-1443R. All reverse transcription reactions were performed on total RNA samples with (+) and without (-) reverse transcriptase and were amplified by PCR in parallel with GT1-7 genomic DNA control and no-template water control (NTC). The size of PCR amplicons was marked by 1 kbp DNA ladder.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Variant Assay, Polymerase Chain Reaction, Synthesized, Amplification

    22) Product Images from "DNA Damage Responses in Human Induced Pluripotent Stem Cells and Embryonic Stem Cells"

    Article Title: DNA Damage Responses in Human Induced Pluripotent Stem Cells and Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013410

    Analysis of DNA damage signaling and repair gene expression in human pluripotent stem cells and differentiated cells. ( A ) Heat map of transcripts analyzed by PCR array. Expression values were normalized over the expression of β-actin and presented as log 10 of relative changes compared to WA07. In comparison to WA07, genes with higher expression are depicted in red, genes with lower expression are depicted in green, and genes with no difference are depicted in black. The genes were grouped according to the pathway in which they participate: ( B ) DNA damage and cell cycle arrest signaling, ( C ) double strand break (DSB) repair, ( D ) mismatch repair (MMR), ( E ) base excision repair (BER), ( F ) nucleotide excision repair (NER). Expression fold differences were calculated using the -ΔΔCt method relative to averaged ES cell lines and normalized using β-actin as endogenous control. The data presented are from three independent experiments, and asterisks label statistical significance as follows: * 0.01
    Figure Legend Snippet: Analysis of DNA damage signaling and repair gene expression in human pluripotent stem cells and differentiated cells. ( A ) Heat map of transcripts analyzed by PCR array. Expression values were normalized over the expression of β-actin and presented as log 10 of relative changes compared to WA07. In comparison to WA07, genes with higher expression are depicted in red, genes with lower expression are depicted in green, and genes with no difference are depicted in black. The genes were grouped according to the pathway in which they participate: ( B ) DNA damage and cell cycle arrest signaling, ( C ) double strand break (DSB) repair, ( D ) mismatch repair (MMR), ( E ) base excision repair (BER), ( F ) nucleotide excision repair (NER). Expression fold differences were calculated using the -ΔΔCt method relative to averaged ES cell lines and normalized using β-actin as endogenous control. The data presented are from three independent experiments, and asterisks label statistical significance as follows: * 0.01

    Techniques Used: Expressing, Polymerase Chain Reaction

    23) Product Images from "A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries"

    Article Title: A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-370

    Comparative digestion of the PCR amplified horse butyrylcholinesterase gene with frequently cleaving REases. TaqII affinity star cleaved 1 μg horse butyrylcholinesterase gene DNA (1841 bp) was electrophoresed on 1.5% agarose/TBE gel. Cleavage was carried out at 65°C for 16 h with 5 μg (40 pmol) of enzyme in 50 μl of reaction volume. Lane M1, Fermentas 1 kb DNA Ladder (selected bands marked); lane M2, Fermentas 100 bp DNA Ladder (selected bands marked); Lane K, undigested DNA; lanes 1–4, DNA digested with TaqII (Methods): lane 1, without SIN and DMSO; lane 2, with SIN, no DMSO; lane 3, no SIN, 20% DMSO; lane 4, with SIN, 20% DMSO; lane 5, DNA digested with HaeIII (5 units); lane 6, DNA digested with CviJI (0.25 unit).
    Figure Legend Snippet: Comparative digestion of the PCR amplified horse butyrylcholinesterase gene with frequently cleaving REases. TaqII affinity star cleaved 1 μg horse butyrylcholinesterase gene DNA (1841 bp) was electrophoresed on 1.5% agarose/TBE gel. Cleavage was carried out at 65°C for 16 h with 5 μg (40 pmol) of enzyme in 50 μl of reaction volume. Lane M1, Fermentas 1 kb DNA Ladder (selected bands marked); lane M2, Fermentas 100 bp DNA Ladder (selected bands marked); Lane K, undigested DNA; lanes 1–4, DNA digested with TaqII (Methods): lane 1, without SIN and DMSO; lane 2, with SIN, no DMSO; lane 3, no SIN, 20% DMSO; lane 4, with SIN, 20% DMSO; lane 5, DNA digested with HaeIII (5 units); lane 6, DNA digested with CviJI (0.25 unit).

    Techniques Used: Polymerase Chain Reaction, Amplification

    Specificity change of TaqII REase in the presence of SIN and DMSO. Relaxed recognition sites were determined by shotgun cloning and sequencing of TaqII restriction fragments obtained in the presence of SIN and DMSO. After digestion DNA was blunted with T4 polymerase and cloned into the SmaI site of pUC19 vector. TaqII – canonical recognition sequences. TaqII*/SIN – variants of SIN induced relaxed recognition sequences.
    Figure Legend Snippet: Specificity change of TaqII REase in the presence of SIN and DMSO. Relaxed recognition sites were determined by shotgun cloning and sequencing of TaqII restriction fragments obtained in the presence of SIN and DMSO. After digestion DNA was blunted with T4 polymerase and cloned into the SmaI site of pUC19 vector. TaqII – canonical recognition sequences. TaqII*/SIN – variants of SIN induced relaxed recognition sequences.

    Techniques Used: Clone Assay, Sequencing, Plasmid Preparation

    PCR fragment DNA substrates. Putative recognition sequence of TaqII is in bold and underlined. Arrows mark the cleavage. The restriction fragments lacking TaqII recognition sequence are in italics. ( A ) PCR fragment without a TaqII site. ( B ) PCR DNA fragment with a single 5′-GACCGA-3′ site.
    Figure Legend Snippet: PCR fragment DNA substrates. Putative recognition sequence of TaqII is in bold and underlined. Arrows mark the cleavage. The restriction fragments lacking TaqII recognition sequence are in italics. ( A ) PCR fragment without a TaqII site. ( B ) PCR DNA fragment with a single 5′-GACCGA-3′ site.

    Techniques Used: Polymerase Chain Reaction, Sequencing

    24) Product Images from "Homology-mediated end-capping as a primary step of sister chromatid fusion in the breakage-fusion-bridge cycles"

    Article Title: Homology-mediated end-capping as a primary step of sister chromatid fusion in the breakage-fusion-bridge cycles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt762

    ( a ) PCR strategy for the amplification of palindromic fusion breakpoints. Lines represent genomic segments. Two PCR primers are indicated by gray arrows. Inverted Alu repeats (or microhomologies) are indicated by a pair of black arrows. ( b ) Ethidium bromide-stained agarose gels after PCR amplification for RDRP2 and ADAMSTL4 junctions. Normal fibroblast IMR90 DNA was used as a negative control. ( c ) DNA sequences of palindromic junctions with Alu inverted repeats. The locations (coordinates of chromosome 1) of inverted Alu repeats and the coordinates (top and bottom) of breakpoints and junction sequences (middle) are shown. Sequence homologies are shown in red. ( d ) DNA sequences of microhomology-mediated palindromic junctions.
    Figure Legend Snippet: ( a ) PCR strategy for the amplification of palindromic fusion breakpoints. Lines represent genomic segments. Two PCR primers are indicated by gray arrows. Inverted Alu repeats (or microhomologies) are indicated by a pair of black arrows. ( b ) Ethidium bromide-stained agarose gels after PCR amplification for RDRP2 and ADAMSTL4 junctions. Normal fibroblast IMR90 DNA was used as a negative control. ( c ) DNA sequences of palindromic junctions with Alu inverted repeats. The locations (coordinates of chromosome 1) of inverted Alu repeats and the coordinates (top and bottom) of breakpoints and junction sequences (middle) are shown. Sequence homologies are shown in red. ( d ) DNA sequences of microhomology-mediated palindromic junctions.

    Techniques Used: Polymerase Chain Reaction, Amplification, Staining, Negative Control, Sequencing

    ( a ) A strategy for enriching DNA from palindromic junctions. DNA is shown by either a black, dotted, or gray line, with the black and dotted lines being complementary to each other. ( b ) Southern analyses of the PCR products using probes in the human genome. The locations of probes (CTSK cen and tel) are shown in ( d ). (i) enrichment is only seen in Colo320DM by probe CTSK cen that is within the palindromic junction, but not by probe CTSK tel, and (ii) probes from a naturally existing DNA inverted repeat at chromosome 19p13.2 (chr19:7049026–7058989 in hg19) showed strong enrichment in both IMR90 and Colo320DM. EtBr, ethidium bromide stained gel. ( c ) Schematic drawings show that in vitro fold-back of palindromic DNA after denaturation/renaturation and subsequent digestion by nuclease S1 (left) can results in the steady increase in read depth (per 1 kb bin) toward a palindromic junction (right). The numbers indicate 1 kb bins on the reference genome. (d) The numbers of GAPF-reads/kb in Colo320DM (closed circles) and IMR90 (open circles) are shown for the 50 kb region of chromosome 1 where the CTSK gene is located. Two genomic features (GC content and SINE elements) within the genomic region are also shown.
    Figure Legend Snippet: ( a ) A strategy for enriching DNA from palindromic junctions. DNA is shown by either a black, dotted, or gray line, with the black and dotted lines being complementary to each other. ( b ) Southern analyses of the PCR products using probes in the human genome. The locations of probes (CTSK cen and tel) are shown in ( d ). (i) enrichment is only seen in Colo320DM by probe CTSK cen that is within the palindromic junction, but not by probe CTSK tel, and (ii) probes from a naturally existing DNA inverted repeat at chromosome 19p13.2 (chr19:7049026–7058989 in hg19) showed strong enrichment in both IMR90 and Colo320DM. EtBr, ethidium bromide stained gel. ( c ) Schematic drawings show that in vitro fold-back of palindromic DNA after denaturation/renaturation and subsequent digestion by nuclease S1 (left) can results in the steady increase in read depth (per 1 kb bin) toward a palindromic junction (right). The numbers indicate 1 kb bins on the reference genome. (d) The numbers of GAPF-reads/kb in Colo320DM (closed circles) and IMR90 (open circles) are shown for the 50 kb region of chromosome 1 where the CTSK gene is located. Two genomic features (GC content and SINE elements) within the genomic region are also shown.

    Techniques Used: Polymerase Chain Reaction, Staining, In Vitro

    25) Product Images from "Deciphering phenotypic variance in different models of DNA-PKcs deficiency."

    Article Title: Deciphering phenotypic variance in different models of DNA-PKcs deficiency.

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2018.10.004

    ATM expression is substantially reduced in cultured cells ablated for DNA-PKcs. (A). DNA-PKcs and ATM protein expression levels were examined by western blotting of whole cell extracts obtained from wild type or SCID ear fibroblasts (left panel), and patient derived fibroblasts (right panel). A non-specific band detected by the ATM antibody (left panel) or Ku86 (right panel) serve as loading controls. (B) ATM protein expression levels were assessed in wild type and DNA-PKcs deficient 293T cells, HCT116 cells, and MO59K/J cells. Protein levels from indicated amounts of whole cell extract were assessed by immunoblotting. A non-specific band detected by the ATM antibody serves as a loading control. (C) ATM expression was assessed from 293T cells ablated for other c-NHEH components as indicated.(D) Expression of the components of the TTT complex were assessed by immunoblotting in wild type 293T cells, or in cells lacking DNA-PKcs, or harboring a kinase inactivating mutation of DNA-PKcs as indicated. White line indicate removal of lanes not presented; figure is derived from a single blot. (E) DNA-PKcs and ATM expression in wild type or DNA-PKcs deficient 293T cells 72 hours after transfection with a DNA-PKcs expression construct or vector alone. A non-specific band detected by the ATM antibody serves as a loading control. (F) GFP and ATM expression levels in wild type or DNA-PKcs deficient 293T cells transfected with either or both GFP-DNA-PKcs or GFP-ATM as indicated.
    Figure Legend Snippet: ATM expression is substantially reduced in cultured cells ablated for DNA-PKcs. (A). DNA-PKcs and ATM protein expression levels were examined by western blotting of whole cell extracts obtained from wild type or SCID ear fibroblasts (left panel), and patient derived fibroblasts (right panel). A non-specific band detected by the ATM antibody (left panel) or Ku86 (right panel) serve as loading controls. (B) ATM protein expression levels were assessed in wild type and DNA-PKcs deficient 293T cells, HCT116 cells, and MO59K/J cells. Protein levels from indicated amounts of whole cell extract were assessed by immunoblotting. A non-specific band detected by the ATM antibody serves as a loading control. (C) ATM expression was assessed from 293T cells ablated for other c-NHEH components as indicated.(D) Expression of the components of the TTT complex were assessed by immunoblotting in wild type 293T cells, or in cells lacking DNA-PKcs, or harboring a kinase inactivating mutation of DNA-PKcs as indicated. White line indicate removal of lanes not presented; figure is derived from a single blot. (E) DNA-PKcs and ATM expression in wild type or DNA-PKcs deficient 293T cells 72 hours after transfection with a DNA-PKcs expression construct or vector alone. A non-specific band detected by the ATM antibody serves as a loading control. (F) GFP and ATM expression levels in wild type or DNA-PKcs deficient 293T cells transfected with either or both GFP-DNA-PKcs or GFP-ATM as indicated.

    Techniques Used: Expressing, Cell Culture, Western Blot, Derivative Assay, Mutagenesis, Transfection, Construct, Plasmid Preparation

    Kinase inactive DNA-PKcs does not alter end-joining deficits in DNA-PKcs deficient cells. (A) The fluorescent substrate ISceRFP/CFP (left panel) was utilized to detect joining of I-Sce1 induced DSBs. Percent recombination of episomal fluorescent I-Sce1 end joining substrate in Sf19 transfectants expressing wild type DNA-PKcs, vector alone, or the D3922 > A mutation. The fluorescent substrate telN-CFP (right panel) was utilized to detect CFP expression from uncut (U) or cut substrate (C), co-transfected with dsRED to assess transfection efficiency. TelN cleavage generates a DNA break with hairpin termini. Percent CFP/RFP is presented. Error bars indicate SEM from three independent experiments. Ns, P value, not significant, in two-tailed unpaired t test. (B) Diagram of region of murine AICDA locus targeted by two gRNAs and position of primers utilized to detect chromosomal deletions. Ethidium bromide staining of PCR amplifications of DNA isolated from Sf19 transfectants expressing wild type DNA-PKcs, vector only, or D3922A mutant DNA-PKcs as indicated. (C) Summary of sequenced joints. P= 0.1916; (not significant) in two-tailed unpaired t test comparing nucleotide loss between sequences from cells expressing vector only versus the D3922 > A mutant.
    Figure Legend Snippet: Kinase inactive DNA-PKcs does not alter end-joining deficits in DNA-PKcs deficient cells. (A) The fluorescent substrate ISceRFP/CFP (left panel) was utilized to detect joining of I-Sce1 induced DSBs. Percent recombination of episomal fluorescent I-Sce1 end joining substrate in Sf19 transfectants expressing wild type DNA-PKcs, vector alone, or the D3922 > A mutation. The fluorescent substrate telN-CFP (right panel) was utilized to detect CFP expression from uncut (U) or cut substrate (C), co-transfected with dsRED to assess transfection efficiency. TelN cleavage generates a DNA break with hairpin termini. Percent CFP/RFP is presented. Error bars indicate SEM from three independent experiments. Ns, P value, not significant, in two-tailed unpaired t test. (B) Diagram of region of murine AICDA locus targeted by two gRNAs and position of primers utilized to detect chromosomal deletions. Ethidium bromide staining of PCR amplifications of DNA isolated from Sf19 transfectants expressing wild type DNA-PKcs, vector only, or D3922A mutant DNA-PKcs as indicated. (C) Summary of sequenced joints. P= 0.1916; (not significant) in two-tailed unpaired t test comparing nucleotide loss between sequences from cells expressing vector only versus the D3922 > A mutant.

    Techniques Used: Expressing, Plasmid Preparation, Mutagenesis, Transfection, Two Tailed Test, Staining, Polymerase Chain Reaction, Isolation

    26) Product Images from "Interaction between amyloid precursor protein and Nogo receptors regulates amyloid deposition"

    Article Title: Interaction between amyloid precursor protein and Nogo receptors regulates amyloid deposition

    Journal: The FASEB Journal

    doi: 10.1096/fj.11-184325

    Validation of genetic deletion of NgR2 in mice. A ) As illustrated, mouse NgR2 is encoded by 3 exons, and the targeting construct was constructed to remove exon 3. After homologous recombination, the original 7.5-kb Bst XI fragment in WT mice was replaced by the 5.8-kb Neo-containing fragment in NgR2-null mice. DNA size markers are 9.4 and 6.5 kb, respectively. Solid line near the last Bst XI site indicates Southern blot probe. B ) Northern blot analysis confirmed that the 2.4-kb NgR2 transcript was abolished in NgR2-deficient mice. β-Actin was used as a loading control.
    Figure Legend Snippet: Validation of genetic deletion of NgR2 in mice. A ) As illustrated, mouse NgR2 is encoded by 3 exons, and the targeting construct was constructed to remove exon 3. After homologous recombination, the original 7.5-kb Bst XI fragment in WT mice was replaced by the 5.8-kb Neo-containing fragment in NgR2-null mice. DNA size markers are 9.4 and 6.5 kb, respectively. Solid line near the last Bst XI site indicates Southern blot probe. B ) Northern blot analysis confirmed that the 2.4-kb NgR2 transcript was abolished in NgR2-deficient mice. β-Actin was used as a loading control.

    Techniques Used: Mouse Assay, Construct, Homologous Recombination, Southern Blot, Northern Blot

    Cell surface protein biotinylation. HEK293 cells were transfected with WT NgR2 expression plasmid DNA or empty vector DNA for 48 h, and surface proteins were labeled by biotinylation. Neutravidin was used to pull down biotinylated surface proteins, followed by Western blotting. Surface biotinylated APP is significantly lowered in NgR2-overexpressing cells, while surface expression of ADAM17 was not significantly affected by NgR2. APP was detected by antibody A8717 and NgR2 by polyclonal NgR2 antibody. Calnexin and actin are shown as loading controls. * P
    Figure Legend Snippet: Cell surface protein biotinylation. HEK293 cells were transfected with WT NgR2 expression plasmid DNA or empty vector DNA for 48 h, and surface proteins were labeled by biotinylation. Neutravidin was used to pull down biotinylated surface proteins, followed by Western blotting. Surface biotinylated APP is significantly lowered in NgR2-overexpressing cells, while surface expression of ADAM17 was not significantly affected by NgR2. APP was detected by antibody A8717 and NgR2 by polyclonal NgR2 antibody. Calnexin and actin are shown as loading controls. * P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Labeling, Western Blot

    27) Product Images from "Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript"

    Article Title: Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2018.0209

    DNA sequence of genomic TNFSF9 gene edited by CRISPR-Cas9 The TNFSF9 genes in genomic DNAs from wild type and mutated HepG2 cells were amplified by PCR. The PCR products were isolated and sequenced as described in “Materials and Methods”. Left panels are chromatograms of the genomic TNFSF9 sequence. On the right are shown the DNA sequences around the region of the mutated triplet in the wildtype and mutant clones. This is one representative of five independent experiments.
    Figure Legend Snippet: DNA sequence of genomic TNFSF9 gene edited by CRISPR-Cas9 The TNFSF9 genes in genomic DNAs from wild type and mutated HepG2 cells were amplified by PCR. The PCR products were isolated and sequenced as described in “Materials and Methods”. Left panels are chromatograms of the genomic TNFSF9 sequence. On the right are shown the DNA sequences around the region of the mutated triplet in the wildtype and mutant clones. This is one representative of five independent experiments.

    Techniques Used: Sequencing, CRISPR, Amplification, Polymerase Chain Reaction, Isolation, Mutagenesis, Clone Assay

    Detection of the sgRNA plasmid in HepG2 clone 9-1 The sgRNA plasmid sequence was amplified by PCR from genomic DNA of wild type and clone 9-1 HepG2 cells, as described in “Materials and Methods”. The PCR products were fractionated on a 2% agarose gel and visualized under UV. This is one representative of three independent experiments.
    Figure Legend Snippet: Detection of the sgRNA plasmid in HepG2 clone 9-1 The sgRNA plasmid sequence was amplified by PCR from genomic DNA of wild type and clone 9-1 HepG2 cells, as described in “Materials and Methods”. The PCR products were fractionated on a 2% agarose gel and visualized under UV. This is one representative of three independent experiments.

    Techniques Used: Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    28) Product Images from "Hypermethylation of p16INK4a in Korean Non-small Cell Lung Cancer Patients"

    Article Title: Hypermethylation of p16INK4a in Korean Non-small Cell Lung Cancer Patients

    Journal: Journal of Korean Medical Science

    doi: 10.3346/jkms.2007.22.S.S32

    Result of serial dilutions to determine the detection limits of the real-time PCR protocol showing the initial DNA amounts used in the amplification.
    Figure Legend Snippet: Result of serial dilutions to determine the detection limits of the real-time PCR protocol showing the initial DNA amounts used in the amplification.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    Results of p16 INK4a gene promoter methylation of normal ( A ) and tumor ( B ) lung tissues by real-time PCR. 2, 3, 5: Normal lung tissues ( A ); 2 * , 3 * , 5 * : tumor lung tissues ( B ); 1, 4, 6: positive control (wi-38); 7: negative control (water); a: no-cut DNA amplification; b: Hpa II-cut DNA amplification; c: Msp I-cut DNA amplification. Delta Rn: the magnitude of the fluorescence signal generated during the PCR at each time point.
    Figure Legend Snippet: Results of p16 INK4a gene promoter methylation of normal ( A ) and tumor ( B ) lung tissues by real-time PCR. 2, 3, 5: Normal lung tissues ( A ); 2 * , 3 * , 5 * : tumor lung tissues ( B ); 1, 4, 6: positive control (wi-38); 7: negative control (water); a: no-cut DNA amplification; b: Hpa II-cut DNA amplification; c: Msp I-cut DNA amplification. Delta Rn: the magnitude of the fluorescence signal generated during the PCR at each time point.

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Amplification, Fluorescence, Generated, Polymerase Chain Reaction

    29) Product Images from "Telomerase activity is required for the telomere G-overhang structure in Trypanosoma brucei"

    Article Title: Telomerase activity is required for the telomere G-overhang structure in Trypanosoma brucei

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16182-y

    Most telomeres in T . brucei WT cells have a 3′ overhang that ends in 5′ TTAGGG 3′. ( a ) A diagram showing the principle of the adaptor ligation assay. Top, a natural chromosome end with a 3′ overhang (left) is ligated with an adaptor (right). The light bar of the adaptor represents the unique oligo and is radiolabeled at its 5′ end. The darker bar of the adaptor represents the common region of all guide oligos. Only when the adaptor matches the telomere end perfectly can the adaptor be ligated to the chromosome end and eventually migrate with the long telomere fragment in agarose gel electrophoresis. Bottom, the sequence of the unique oligo and seven different guide oligos are shown. The T . brucei genomic DNA from BF ( b ) and PF ( c ) WT cells (either treated with or without Exo T) was ligated to radioisotope-labeled different adaptors (TG1–6 and TGNS, as indicated on top of each lane), digested with AluI and MboI, and separated by agarose gel electrophoresis. The ethidium bromide-stained gel is shown on the left and the image of the gel exposed to a phosphorimager (exposed) is shown on the right. 1 kb DNA ladder (ThermoFisher) was used as a molecular weight marker. ( d ) Quantification of the adaptor ligation results in both BF and PF WT cells. Average values are calculated from five (BF) or three (PF) independent experiments. Error bars represent standard deviation.
    Figure Legend Snippet: Most telomeres in T . brucei WT cells have a 3′ overhang that ends in 5′ TTAGGG 3′. ( a ) A diagram showing the principle of the adaptor ligation assay. Top, a natural chromosome end with a 3′ overhang (left) is ligated with an adaptor (right). The light bar of the adaptor represents the unique oligo and is radiolabeled at its 5′ end. The darker bar of the adaptor represents the common region of all guide oligos. Only when the adaptor matches the telomere end perfectly can the adaptor be ligated to the chromosome end and eventually migrate with the long telomere fragment in agarose gel electrophoresis. Bottom, the sequence of the unique oligo and seven different guide oligos are shown. The T . brucei genomic DNA from BF ( b ) and PF ( c ) WT cells (either treated with or without Exo T) was ligated to radioisotope-labeled different adaptors (TG1–6 and TGNS, as indicated on top of each lane), digested with AluI and MboI, and separated by agarose gel electrophoresis. The ethidium bromide-stained gel is shown on the left and the image of the gel exposed to a phosphorimager (exposed) is shown on the right. 1 kb DNA ladder (ThermoFisher) was used as a molecular weight marker. ( d ) Quantification of the adaptor ligation results in both BF and PF WT cells. Average values are calculated from five (BF) or three (PF) independent experiments. Error bars represent standard deviation.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Sequencing, Labeling, Staining, Molecular Weight, Marker, Standard Deviation

    Telomeres in T . brucei cells prefer to have a 5′ end with a sequence of 5′ CCTAAC 3′. ( a ) A diagram showing the principle of the Telomere Oligo Ligation-mediated PCR (TOLP) assay. Seven different telomere guide oligos are separately ligated to intact genomic DNA. Only when the oligo is aligned immediately next to the 5′ end of the telomere can the oligo be ligated to the chromosome DNA. Subsequently the ligation products are amplified by PCR using a forward primer with the sequence of (TTAGGG) 3 and a backward primer with the sequence identical to the common regions of all guide oligos. The PCR products were then separated by agarose gel electrophoresis followed by Southern analysis using a telomeric probe. ( b ) TOLP was performed using genomic DNA isolated from WT BF cells that was treated with or without T7 exonuclease. The ethidium bromide-stained gel is shown on the top, and the Southern hybridization result using a telomeric probe is shown on the bottom. ( c ) Quantification of the TOLP signals from different guide oligos is shown. Average is calculated from three independent experiments. Error bars represent standard deviation.
    Figure Legend Snippet: Telomeres in T . brucei cells prefer to have a 5′ end with a sequence of 5′ CCTAAC 3′. ( a ) A diagram showing the principle of the Telomere Oligo Ligation-mediated PCR (TOLP) assay. Seven different telomere guide oligos are separately ligated to intact genomic DNA. Only when the oligo is aligned immediately next to the 5′ end of the telomere can the oligo be ligated to the chromosome DNA. Subsequently the ligation products are amplified by PCR using a forward primer with the sequence of (TTAGGG) 3 and a backward primer with the sequence identical to the common regions of all guide oligos. The PCR products were then separated by agarose gel electrophoresis followed by Southern analysis using a telomeric probe. ( b ) TOLP was performed using genomic DNA isolated from WT BF cells that was treated with or without T7 exonuclease. The ethidium bromide-stained gel is shown on the top, and the Southern hybridization result using a telomeric probe is shown on the bottom. ( c ) Quantification of the TOLP signals from different guide oligos is shown. Average is calculated from three independent experiments. Error bars represent standard deviation.

    Techniques Used: Sequencing, Ligation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Isolation, Staining, Hybridization, Standard Deviation

    30) Product Images from "Genome-wide identification of genic and intergenic neuronal DNA regions bound by Tau protein under physiological and stress conditions"

    Article Title: Genome-wide identification of genic and intergenic neuronal DNA regions bound by Tau protein under physiological and stress conditions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky929

    Identification of Tau-interacting regions under physiological conditions by ChIP-on-chip. ( A ) Schematic diagram describing the different steps of the analysis of results of ChIP-on-chip assays carried out after immunoprecipitation of the genome of murine neurons with Tau1 and anti-NSs antibodies. Analysis was carried out using the PGS software that uses MAT algorithm to detect significantly enriched DNA sequences. ( B ) Principal component analysis (PCA) plot that separates samples according to abundance variation showing that the Tau1 samples cluster tightly (little variation) compared to non-specific anti-NSs samples that displayed strong variation. ( C ) General characteristics of DNA regions identified as significantly enriched in Tau1 compared to anti-NSs samples, considered as Tau-interacting regions. The P -value of the region corresponds to the empirical P -value (as determined using a two-way ANOVA test) of the most significant probe MAT score included within this region and the MAT score of the region is the maximum probe MAT score for this region. The P -values and MAT scores of Tau-interacting regions are indicated in Supplementary Table S1 . Positive MAT scores: Tau1 enriched compared to anti-NSs.
    Figure Legend Snippet: Identification of Tau-interacting regions under physiological conditions by ChIP-on-chip. ( A ) Schematic diagram describing the different steps of the analysis of results of ChIP-on-chip assays carried out after immunoprecipitation of the genome of murine neurons with Tau1 and anti-NSs antibodies. Analysis was carried out using the PGS software that uses MAT algorithm to detect significantly enriched DNA sequences. ( B ) Principal component analysis (PCA) plot that separates samples according to abundance variation showing that the Tau1 samples cluster tightly (little variation) compared to non-specific anti-NSs samples that displayed strong variation. ( C ) General characteristics of DNA regions identified as significantly enriched in Tau1 compared to anti-NSs samples, considered as Tau-interacting regions. The P -value of the region corresponds to the empirical P -value (as determined using a two-way ANOVA test) of the most significant probe MAT score included within this region and the MAT score of the region is the maximum probe MAT score for this region. The P -values and MAT scores of Tau-interacting regions are indicated in Supplementary Table S1 . Positive MAT scores: Tau1 enriched compared to anti-NSs.

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Software

    General characteristics of Tau-interacting regions identified under HS conditions. General characteristics of the DNA sequences identified through ChIP-on-chip technique, Tau1 versus anti-NSs (NSs) antibody, as Tau-interacting regions under HS conditions. ( A ) Principal component analysis (PCA) plot that separates samples according to abundance variation showing that under HS conditions Tau1 samples cluster tightly (little variation) compared to non-specific anti-NSs (NSs) samples that displayed strong variation. ( B ) General characteristics of DNA regions identified as significantly enriched in Tau1 samples under HS conditions, considered as Tau-interacting regions. The P -value of the region corresponds to the empirical P -value (as determined using a two-way ANOVA test) of the most significant probe MAT score included within this region and the MAT score of the region is the maximum probe MAT score for this region. The P -values and MAT scores of Tau-interacting regions are indicated in Supplementary Table S3 . ( C ) Distribution among the chromosomes of the DNA probes bound by Tau protein, either genic or intergenic, under physiological (control) and HS conditions expressed as a percentage of the total number of probes per chromosome present in the array. D ) Number of genic and intergenic Tau-interacting regions overlapping with DNA regions coding for lncRNA under HS conditions.
    Figure Legend Snippet: General characteristics of Tau-interacting regions identified under HS conditions. General characteristics of the DNA sequences identified through ChIP-on-chip technique, Tau1 versus anti-NSs (NSs) antibody, as Tau-interacting regions under HS conditions. ( A ) Principal component analysis (PCA) plot that separates samples according to abundance variation showing that under HS conditions Tau1 samples cluster tightly (little variation) compared to non-specific anti-NSs (NSs) samples that displayed strong variation. ( B ) General characteristics of DNA regions identified as significantly enriched in Tau1 samples under HS conditions, considered as Tau-interacting regions. The P -value of the region corresponds to the empirical P -value (as determined using a two-way ANOVA test) of the most significant probe MAT score included within this region and the MAT score of the region is the maximum probe MAT score for this region. The P -values and MAT scores of Tau-interacting regions are indicated in Supplementary Table S3 . ( C ) Distribution among the chromosomes of the DNA probes bound by Tau protein, either genic or intergenic, under physiological (control) and HS conditions expressed as a percentage of the total number of probes per chromosome present in the array. D ) Number of genic and intergenic Tau-interacting regions overlapping with DNA regions coding for lncRNA under HS conditions.

    Techniques Used: Chromatin Immunoprecipitation

    31) Product Images from "Amplicon-Based RNA Interference Targeting V2 Gene of Cotton Leaf Curl Kokhran Virus-Burewala Strain Can Provide Resistance in Transgenic Cotton Plants"

    Article Title: Amplicon-Based RNA Interference Targeting V2 Gene of Cotton Leaf Curl Kokhran Virus-Burewala Strain Can Provide Resistance in Transgenic Cotton Plants

    Journal: Molecular Biotechnology

    doi: 10.1007/s12033-016-9980-8

    a and b Southern Blot Analysis and Restriction digestion gel of Southern for RNAi in transgenic Cotton plant Lane 1 1 Kb DNA Ladder, Lane 2 MC2-8B transgenic cotton plant of MNH 786 variety, Lane 3 VC2-11 transgenic cotton plant of variety VH-289, Lane 4 Non-transgenic cotton control plant
    Figure Legend Snippet: a and b Southern Blot Analysis and Restriction digestion gel of Southern for RNAi in transgenic Cotton plant Lane 1 1 Kb DNA Ladder, Lane 2 MC2-8B transgenic cotton plant of MNH 786 variety, Lane 3 VC2-11 transgenic cotton plant of variety VH-289, Lane 4 Non-transgenic cotton control plant

    Techniques Used: Southern Blot, Transgenic Assay

    PCR analysis of putative transgenic plants (VH-289 MNH-786) L 50 bp DNA ladder; − ve the non-transgenic plant, + ve plasmid construct was used as positive control, V 1– V 6 putative transgenic plants of VH-289, M 1– M 6 putative transgenic plants of MNH-786
    Figure Legend Snippet: PCR analysis of putative transgenic plants (VH-289 MNH-786) L 50 bp DNA ladder; − ve the non-transgenic plant, + ve plasmid construct was used as positive control, V 1– V 6 putative transgenic plants of VH-289, M 1– M 6 putative transgenic plants of MNH-786

    Techniques Used: Polymerase Chain Reaction, Transgenic Assay, Plasmid Preparation, Construct, Positive Control

    32) Product Images from "Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens"

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02224

    Bland–Altman bias plots for two different quantitative MTBC DNA real-time PCR assays. Five serial dilutions of M. bovis BCG (A) and M. tuberculosis mc 2 7000 (B) strain were tested for MTBC DNA quantification by IS6110 and senX3 PCR assays. The mean bias was determined to be 4.026 and 7.455-cycle threshold for M. bovis BCG and M. tuberculosis mc 2 7000, respectively.
    Figure Legend Snippet: Bland–Altman bias plots for two different quantitative MTBC DNA real-time PCR assays. Five serial dilutions of M. bovis BCG (A) and M. tuberculosis mc 2 7000 (B) strain were tested for MTBC DNA quantification by IS6110 and senX3 PCR assays. The mean bias was determined to be 4.026 and 7.455-cycle threshold for M. bovis BCG and M. tuberculosis mc 2 7000, respectively.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Comparison of DNA extraction protocols in spiked sputum samples. The M. tuberculosis mc 2 7000 stock suspension was diluted and used to spike negative sputum samples. Box plots with C T median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ® method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.
    Figure Legend Snippet: Comparison of DNA extraction protocols in spiked sputum samples. The M. tuberculosis mc 2 7000 stock suspension was diluted and used to spike negative sputum samples. Box plots with C T median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ® method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.

    Techniques Used: DNA Extraction, Sonication

    33) Product Images from "Antiretroviral Therapy-Induced Mitochondrial Toxicity: Potential Mechanisms Beyond Polymerase-γ Inhibition"

    Article Title: Antiretroviral Therapy-Induced Mitochondrial Toxicity: Potential Mechanisms Beyond Polymerase-γ Inhibition

    Journal: Clinical pharmacology and therapeutics

    doi: 10.1038/clpt.2014.64

    Figure 4A. Mitochondrial DNA copy number among study groups. A fragment of D-Loop of the mitochondrial DNA was amplified in duplicate in two independent experiments using quantitative RT-PCR. The absolute mtDNA copy number was calculated using serial dilutions of plasmid with known copy numbers of mtDNA (D-loop). Data are represented as box-and-stem plots, in which boxes represent the median values and the second and third quartiles, and stems extend to the 10 th and 90 th percentiles. Different superscripts * , ** , *** indicate significant differences at p≤0.05, ≤0.01, and ≤0.001, respectively. NS, no significance. Figure 4B. Differences in mRNA expression of Pol-γ among study groups. Quantitative real-time PCR was performed using RNA extracted from PBMCs to determine the mRNA expression of Pol-γ compared with GAPDH. The mRNA expression of Pol-γ was calculated as an expression index (EI), where EI = 1000 × 2 −ΔCt , where ΔCt = Ct pol-γ − Ct GAPDH . Data are represented as box-and-stem plots, in which boxes represent the median values and the second and third quartiles, and stems extend to the 10 th and 90 th percentiles. Different superscripts * , ** , *** indicate significant differences at p≤0.05, ≤0.01, and ≤0.001, respectively. NS, no significance.
    Figure Legend Snippet: Figure 4A. Mitochondrial DNA copy number among study groups. A fragment of D-Loop of the mitochondrial DNA was amplified in duplicate in two independent experiments using quantitative RT-PCR. The absolute mtDNA copy number was calculated using serial dilutions of plasmid with known copy numbers of mtDNA (D-loop). Data are represented as box-and-stem plots, in which boxes represent the median values and the second and third quartiles, and stems extend to the 10 th and 90 th percentiles. Different superscripts * , ** , *** indicate significant differences at p≤0.05, ≤0.01, and ≤0.001, respectively. NS, no significance. Figure 4B. Differences in mRNA expression of Pol-γ among study groups. Quantitative real-time PCR was performed using RNA extracted from PBMCs to determine the mRNA expression of Pol-γ compared with GAPDH. The mRNA expression of Pol-γ was calculated as an expression index (EI), where EI = 1000 × 2 −ΔCt , where ΔCt = Ct pol-γ − Ct GAPDH . Data are represented as box-and-stem plots, in which boxes represent the median values and the second and third quartiles, and stems extend to the 10 th and 90 th percentiles. Different superscripts * , ** , *** indicate significant differences at p≤0.05, ≤0.01, and ≤0.001, respectively. NS, no significance.

    Techniques Used: Amplification, Quantitative RT-PCR, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction

    Potential mechanisms of ART-induced mitochondrial toxicity. A. Biosynthesis of endogenous nucleotides under normal conditions: Deoxyribonucleotise (dN) is transported into the cell through nucleoside transporter (NT). dN is phosphorylated to deoxyribonucleotide triphosphate (dNTP) in a step-wise fashion using deoxyribonucleoside kinases such thymidine kinase (TK1) and deoxycytidine kinase (dCK); nucleoside monophosphate kinases (NMPKs); and nucleoside diphosphate kinases (NDPKs): the salvage pathway. dNTP is also produced via the de novo pathway through the reduction of ribonucleotide diphosphate (NDP) to deoxyribonucleotide (dNDP) catalyzed by ribonucleotide reductase (RR). dNTP is used for the synthesis of both nuclear and mitochondrial DNA. Mitochondrial dNTP arises from both an import from the cytosol and synthesis in the mitochondrial through the salvage pathway. Mitochondrial DNA contains genes that encodes for essential proteins of the electronic transport chain (ETC). Monophosphates of both endogenous and nucleoside analog nucleotides can be efflux by ATP-Binding Cassette (ABC) proteins in cell membrane. B. Biosynthesis of nucleotides during ART-induced mitochondrial toxicity. Nucleoside analogs (NA) are metabolized in a step-wise fashion catalyze by the enzymes of the salvage pathway in the cytosol and mitochondria. The metabolism and usage of nucleoside analog triphosphates (NA-PPP) with time lead to competitive and feedback inhibition of the metabolism of endogenous nucleotides. Decreased synthesis of ribonucleotides (NMP, ribonucleotide monophosphate; NDP, ribonucleotide diphosphate) and dNTPs result in decreased DNA synthesis in the nucleus and mitochondrial; indicated by fading of the endogenous pathway. Analog triphosphate (NA-PPP) incorporation in DNA results inhibition of nuclear and mitochondrial DNA synthesis. Compensatory attempt at increasing DNA synthesis results in increased expression of deoxyribonucleoside kinases (TK1/2, dCK). The excess of analog metabolites could contribute to five potential mechanisms of mitochondrial toxicity numbered in the diagram: [1] Competitive inhibition of nucleotide metabolism resulting in decreased DNA synthesis; [2] Inhibition of Pol-γ by NA-PPP; [3] Compensatory proliferation of mtDNA; [4] Direct effect of ART on mitochondrial DNA replication and translation processes; [5] Production of reactive oxygen species (ROS).
    Figure Legend Snippet: Potential mechanisms of ART-induced mitochondrial toxicity. A. Biosynthesis of endogenous nucleotides under normal conditions: Deoxyribonucleotise (dN) is transported into the cell through nucleoside transporter (NT). dN is phosphorylated to deoxyribonucleotide triphosphate (dNTP) in a step-wise fashion using deoxyribonucleoside kinases such thymidine kinase (TK1) and deoxycytidine kinase (dCK); nucleoside monophosphate kinases (NMPKs); and nucleoside diphosphate kinases (NDPKs): the salvage pathway. dNTP is also produced via the de novo pathway through the reduction of ribonucleotide diphosphate (NDP) to deoxyribonucleotide (dNDP) catalyzed by ribonucleotide reductase (RR). dNTP is used for the synthesis of both nuclear and mitochondrial DNA. Mitochondrial dNTP arises from both an import from the cytosol and synthesis in the mitochondrial through the salvage pathway. Mitochondrial DNA contains genes that encodes for essential proteins of the electronic transport chain (ETC). Monophosphates of both endogenous and nucleoside analog nucleotides can be efflux by ATP-Binding Cassette (ABC) proteins in cell membrane. B. Biosynthesis of nucleotides during ART-induced mitochondrial toxicity. Nucleoside analogs (NA) are metabolized in a step-wise fashion catalyze by the enzymes of the salvage pathway in the cytosol and mitochondria. The metabolism and usage of nucleoside analog triphosphates (NA-PPP) with time lead to competitive and feedback inhibition of the metabolism of endogenous nucleotides. Decreased synthesis of ribonucleotides (NMP, ribonucleotide monophosphate; NDP, ribonucleotide diphosphate) and dNTPs result in decreased DNA synthesis in the nucleus and mitochondrial; indicated by fading of the endogenous pathway. Analog triphosphate (NA-PPP) incorporation in DNA results inhibition of nuclear and mitochondrial DNA synthesis. Compensatory attempt at increasing DNA synthesis results in increased expression of deoxyribonucleoside kinases (TK1/2, dCK). The excess of analog metabolites could contribute to five potential mechanisms of mitochondrial toxicity numbered in the diagram: [1] Competitive inhibition of nucleotide metabolism resulting in decreased DNA synthesis; [2] Inhibition of Pol-γ by NA-PPP; [3] Compensatory proliferation of mtDNA; [4] Direct effect of ART on mitochondrial DNA replication and translation processes; [5] Production of reactive oxygen species (ROS).

    Techniques Used: Produced, Binding Assay, Inhibition, DNA Synthesis, Expressing

    34) Product Images from "Overexpression of Bacterial mtlD Gene in Peanut Improves Drought Tolerance through Accumulation of Mannitol"

    Article Title: Overexpression of Bacterial mtlD Gene in Peanut Improves Drought Tolerance through Accumulation of Mannitol

    Journal: The Scientific World Journal

    doi: 10.1155/2014/125967

    Molecular characterization of mtlD peanut transformants. (a) PCR amplification using mtlD gene-specific primers (expected size 600 bp); (b) PCR amplification of transformants using npt II gene specific primers (expected size 750 bp), where lane N: negative control; lane P: positive control (pCAMBIA1380 plasmid DNA); and lanes 1–8: transgenic lines (MTD1 to MTD8); (c) Southern blot analysis of transgenic (T 0 ) and nontransformed peanut lines. Where lanes 1–4: DNA from transgenic lines, lane 5: DNA from nontransformed line (cv. GG 20, −ve control), and lane 6: plasmid DNA (+ve control); (d) detection of mtlD gene transcription in transgenic plants using RT-PCR. Lane N = nontransformed line; lanes 1–8 = transgenic lines (MTD1 to MTD8), bottom row: 18SrRNA as internal control.
    Figure Legend Snippet: Molecular characterization of mtlD peanut transformants. (a) PCR amplification using mtlD gene-specific primers (expected size 600 bp); (b) PCR amplification of transformants using npt II gene specific primers (expected size 750 bp), where lane N: negative control; lane P: positive control (pCAMBIA1380 plasmid DNA); and lanes 1–8: transgenic lines (MTD1 to MTD8); (c) Southern blot analysis of transgenic (T 0 ) and nontransformed peanut lines. Where lanes 1–4: DNA from transgenic lines, lane 5: DNA from nontransformed line (cv. GG 20, −ve control), and lane 6: plasmid DNA (+ve control); (d) detection of mtlD gene transcription in transgenic plants using RT-PCR. Lane N = nontransformed line; lanes 1–8 = transgenic lines (MTD1 to MTD8), bottom row: 18SrRNA as internal control.

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Plasmid Preparation, Transgenic Assay, Southern Blot, Reverse Transcription Polymerase Chain Reaction

    Schematic representation of the T-DNA region of pCAMBIA 1380 binary plasmid used for transformation of deembryonated cotyledons with Agrobacterium tumefaciens strain LBA 4404. The position of the primers used in PCR assays is shown by arrows on top of the mtlD gene. LB, left T-DNA border sequence; RB, right border sequence; 35S, CaMV35S promoter; and mtlD , mannitol-1-phosphate dehydrogenase.
    Figure Legend Snippet: Schematic representation of the T-DNA region of pCAMBIA 1380 binary plasmid used for transformation of deembryonated cotyledons with Agrobacterium tumefaciens strain LBA 4404. The position of the primers used in PCR assays is shown by arrows on top of the mtlD gene. LB, left T-DNA border sequence; RB, right border sequence; 35S, CaMV35S promoter; and mtlD , mannitol-1-phosphate dehydrogenase.

    Techniques Used: Plasmid Preparation, Transformation Assay, Polymerase Chain Reaction, Sequencing

    35) Product Images from "Genetic Authentication of Gardenia jasminoides Ellis var. grandiflora Nakai by Improved RAPD-Derived DNA Markers"

    Article Title: Genetic Authentication of Gardenia jasminoides Ellis var. grandiflora Nakai by Improved RAPD-Derived DNA Markers

    Journal: Molecules

    doi: 10.3390/molecules201119687

    Development of stable RAPD-sequence-characterized amplified region (SCAR) markers for ZZH11, ZZH31, ZZH41 and ZZH51. ( A ) A SCAR marker ZZH11; ( B ) A SCAR marker ZZH31; ( C ) A SCAR marker ZZH41; ( D ) A SCAR marker ZZH51. Lanes 1~6 indicate the different samples of G. jasminoides Ellis var. grandiflora Nakai listed in Table 1 . Lanes 7 and 8 are two samples of Lonicera japonica from Hunan and Hubei; Lane 9 is one sample of Penthorum chinense ; Lane 10 is one sample of Ganoderma lucidum ; Lanes 11 and 12 are two samples of Litchi chinesis from Guangdong and Sichuan; Lanes 13 and 14 are two samples of Dimocarpus Longan from Fujian and Sichuan; Lane 15 is one sample of Dimocarpus confinis from Guangxi; Lanes 16 and 17 are two samples of Ginkgo Biloba from Sichuan and Hunan; Lanes 18 and 19 are two samples of Angelica sinensis from Sichuan and Gansu; Lane 20 is one sample of Gastrodia elata ; Lanes 21 and 22 are two samples of Canarium album Guangdong and Sichuan; Lane 23 is negative control without DNA. The blue arrows indicate expected PCR products in size. Lane M indicates the DNA molecular weight marker DL2000.
    Figure Legend Snippet: Development of stable RAPD-sequence-characterized amplified region (SCAR) markers for ZZH11, ZZH31, ZZH41 and ZZH51. ( A ) A SCAR marker ZZH11; ( B ) A SCAR marker ZZH31; ( C ) A SCAR marker ZZH41; ( D ) A SCAR marker ZZH51. Lanes 1~6 indicate the different samples of G. jasminoides Ellis var. grandiflora Nakai listed in Table 1 . Lanes 7 and 8 are two samples of Lonicera japonica from Hunan and Hubei; Lane 9 is one sample of Penthorum chinense ; Lane 10 is one sample of Ganoderma lucidum ; Lanes 11 and 12 are two samples of Litchi chinesis from Guangdong and Sichuan; Lanes 13 and 14 are two samples of Dimocarpus Longan from Fujian and Sichuan; Lane 15 is one sample of Dimocarpus confinis from Guangxi; Lanes 16 and 17 are two samples of Ginkgo Biloba from Sichuan and Hunan; Lanes 18 and 19 are two samples of Angelica sinensis from Sichuan and Gansu; Lane 20 is one sample of Gastrodia elata ; Lanes 21 and 22 are two samples of Canarium album Guangdong and Sichuan; Lane 23 is negative control without DNA. The blue arrows indicate expected PCR products in size. Lane M indicates the DNA molecular weight marker DL2000.

    Techniques Used: Sequencing, Amplification, Marker, Negative Control, Polymerase Chain Reaction, Molecular Weight

    DNA cloning and identification of positive clones. ( A ) Clone identification of RAPD fragment 1. Lanes 6~12 indicate different clones; ( B ) Clone identification of RAPD fragment 3. Lanes 31~39 indicate different clones; ( C ) Clones identification of RAPD fragment 4. Lanes 41~44 indicate different clones; ( D ) Clone identification of RAPD fragment 5. Lanes 51~55 indicate different clones. The blue arrows indicate positive PCR products. Clones ZZH11, ZZH31, ZZH41 and ZZH51 in blue colors were sequencing. The blue arrows indicate expected PCR bands in size of different clones. Lane M indicates the DNA molecular weight marker DL2000 with the fragment size (bp).
    Figure Legend Snippet: DNA cloning and identification of positive clones. ( A ) Clone identification of RAPD fragment 1. Lanes 6~12 indicate different clones; ( B ) Clone identification of RAPD fragment 3. Lanes 31~39 indicate different clones; ( C ) Clones identification of RAPD fragment 4. Lanes 41~44 indicate different clones; ( D ) Clone identification of RAPD fragment 5. Lanes 51~55 indicate different clones. The blue arrows indicate positive PCR products. Clones ZZH11, ZZH31, ZZH41 and ZZH51 in blue colors were sequencing. The blue arrows indicate expected PCR bands in size of different clones. Lane M indicates the DNA molecular weight marker DL2000 with the fragment size (bp).

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Sequencing, Molecular Weight, Marker

    36) Product Images from "Submillimolar levels of calcium regulates DNA structure at the dinucleotide repeat (TG/AC)n"

    Article Title: Submillimolar levels of calcium regulates DNA structure at the dinucleotide repeat (TG/AC)n

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Ca 2+ /CaM-PK II autophosphorylation assay. The activity of the Ca 2+ /CaM-PK II is indicated by its phosphorylated 50- and 60-kDa subunits (I and II) in the assay. Basal activities are shown in the absence of DNA (lanes 2 and 3). Addition of 300 μM exogenous calcium increased phosphorylation (lane 4). Plasmid DNA containing residual calcium (dialyzed but no BAPTA treatment) donated calcium as indicated by increased autophosphorylation (lane 5), whereas calcium-free DNA completely inhibited autophosphorylation (lane 6). Exogenous calcium prevented the inhibitory effect of calcium-free plasmid DNA (lane 7). The specific inhibitor of CaM [CaM kinase II(290–309) CaM antagonist] completely blocked autophosphorylation (band I) (lane 8); addition of BSA (lane B) did not alter basal activity (lane 9); addition of calcium-free rat genomic DNA (lane G) also inhibited autophosphorylation (lane 10). The first band from the top in lane 9 is the result of BSA phosphorylation. Bands I and II indicate the two subunits of the enzyme; numbers indicate the molecular mass of the protein standards in kDa.
    Figure Legend Snippet: Ca 2+ /CaM-PK II autophosphorylation assay. The activity of the Ca 2+ /CaM-PK II is indicated by its phosphorylated 50- and 60-kDa subunits (I and II) in the assay. Basal activities are shown in the absence of DNA (lanes 2 and 3). Addition of 300 μM exogenous calcium increased phosphorylation (lane 4). Plasmid DNA containing residual calcium (dialyzed but no BAPTA treatment) donated calcium as indicated by increased autophosphorylation (lane 5), whereas calcium-free DNA completely inhibited autophosphorylation (lane 6). Exogenous calcium prevented the inhibitory effect of calcium-free plasmid DNA (lane 7). The specific inhibitor of CaM [CaM kinase II(290–309) CaM antagonist] completely blocked autophosphorylation (band I) (lane 8); addition of BSA (lane B) did not alter basal activity (lane 9); addition of calcium-free rat genomic DNA (lane G) also inhibited autophosphorylation (lane 10). The first band from the top in lane 9 is the result of BSA phosphorylation. Bands I and II indicate the two subunits of the enzyme; numbers indicate the molecular mass of the protein standards in kDa.

    Techniques Used: Chick Chorioallantoic Membrane Assay, Activity Assay, Plasmid Preparation

    Calcium-binding assay. ( A ) Representative PhosphorImager pictures of Zeta-Probe membranes with immobilized identical slots of CaM, BSA, circular plasmid DNA [(TG) 28 DNAcir] and linearized [(TG) 28 DNAlin] plasmid DNA containing the (TG) 28 repeat, and circular plasmid DNA from which the (TG) 28 repeat was deleted (DNAcirc). All membranes were incubated with 45 CaCl 2 followed by the following treatments: basal binding (Basal), remaining binding after wash with 50 μM EDTA (+EDTA), remaining binding after wash with 1 mM BAPTA (+BAPTA), remaining binding after wash with 1 mM unlabeled CaCl 2 (+cold CaCl 2 ), binding after washing out unlabeled CaCl 2 followed by a reincubation with 50 μM 45 CaCl 2 (Reverse). ( B ) Graphic expression of 45 CaCl 2 binding. Data points are the mean ± SEM of relative activities of the different treatment groups normalized to nonspecific background binding ( n = 3).
    Figure Legend Snippet: Calcium-binding assay. ( A ) Representative PhosphorImager pictures of Zeta-Probe membranes with immobilized identical slots of CaM, BSA, circular plasmid DNA [(TG) 28 DNAcir] and linearized [(TG) 28 DNAlin] plasmid DNA containing the (TG) 28 repeat, and circular plasmid DNA from which the (TG) 28 repeat was deleted (DNAcirc). All membranes were incubated with 45 CaCl 2 followed by the following treatments: basal binding (Basal), remaining binding after wash with 50 μM EDTA (+EDTA), remaining binding after wash with 1 mM BAPTA (+BAPTA), remaining binding after wash with 1 mM unlabeled CaCl 2 (+cold CaCl 2 ), binding after washing out unlabeled CaCl 2 followed by a reincubation with 50 μM 45 CaCl 2 (Reverse). ( B ) Graphic expression of 45 CaCl 2 binding. Data points are the mean ± SEM of relative activities of the different treatment groups normalized to nonspecific background binding ( n = 3).

    Techniques Used: Binding Assay, Chick Chorioallantoic Membrane Assay, Plasmid Preparation, Incubation, Expressing

    37) Product Images from "Poly (ADP-ribose) prevents pathological phase separation of TDP-43 by promoting liquid demixing and stress-granule localization"

    Article Title: Poly (ADP-ribose) prevents pathological phase separation of TDP-43 by promoting liquid demixing and stress-granule localization

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.07.002

    The PAR-binding region and the N-terminus are required for TDP-43 LLPS in vitro . (A) The NLS of human TDP-43 and the Drosophila homologue TAR DNA-binding protein-43 homolog (TBPH), share 42.9% identity. The protein domains of TDP-43-WT, -Q331K, ΔPBM, -C35 (amino acids 85–414) and -C25 (amino acids 176–414) are shown. NLS: nuclear localization sequence, PBM: PAR-binding motif, RRM: RNA recognition motif, NES: nuclear export sequence and PrLD: prion-like domain. (B) LLPS of 10 μM protein in 150mM NaCl. SUMO-TDP-43-WT underwent LLPS but SUMOTDP-43-ΔPBM and SUMO-TDP-43-C35 formed irregular-solid structures that did not fuse. SUMO-TDP-43-C25 formed fibrillar aggregates. TDP-43-Q331K formed liquid droplets and the occasional irregular-solid structure. Performed three independent times with two protein preparations of TDP-43-WT, C35 and C25 and twice from one protein preparation of TDP-43-Q331K. (C) The presence of liquid droplets, no liquid droplet, irregular solid structures, both liquid droplets and irregular solid structures and fibrillar aggregates is plotted. (D) LLPS at 5 μM protein and 50mM NaCl. PAR at 5 μM equivalents to ADP-ribose promoted LLPS of TDP-43-WT and did not promote LLPS of TDP-43-ΔPBM, TDP-43-C35, and TDP-43-C25. PAR promoted the formation of irregular-solid structures of TDP-43Q331K. Performed three independent times with two independent protein preparations. (E) The presence of liquid droplets, no liquid droplet, irregular-solid structures and fibrillar aggregates is plotted for each TDP-43 variant in the absence and presence of PAR.All at room temperature.
    Figure Legend Snippet: The PAR-binding region and the N-terminus are required for TDP-43 LLPS in vitro . (A) The NLS of human TDP-43 and the Drosophila homologue TAR DNA-binding protein-43 homolog (TBPH), share 42.9% identity. The protein domains of TDP-43-WT, -Q331K, ΔPBM, -C35 (amino acids 85–414) and -C25 (amino acids 176–414) are shown. NLS: nuclear localization sequence, PBM: PAR-binding motif, RRM: RNA recognition motif, NES: nuclear export sequence and PrLD: prion-like domain. (B) LLPS of 10 μM protein in 150mM NaCl. SUMO-TDP-43-WT underwent LLPS but SUMOTDP-43-ΔPBM and SUMO-TDP-43-C35 formed irregular-solid structures that did not fuse. SUMO-TDP-43-C25 formed fibrillar aggregates. TDP-43-Q331K formed liquid droplets and the occasional irregular-solid structure. Performed three independent times with two protein preparations of TDP-43-WT, C35 and C25 and twice from one protein preparation of TDP-43-Q331K. (C) The presence of liquid droplets, no liquid droplet, irregular solid structures, both liquid droplets and irregular solid structures and fibrillar aggregates is plotted. (D) LLPS at 5 μM protein and 50mM NaCl. PAR at 5 μM equivalents to ADP-ribose promoted LLPS of TDP-43-WT and did not promote LLPS of TDP-43-ΔPBM, TDP-43-C35, and TDP-43-C25. PAR promoted the formation of irregular-solid structures of TDP-43Q331K. Performed three independent times with two independent protein preparations. (E) The presence of liquid droplets, no liquid droplet, irregular-solid structures and fibrillar aggregates is plotted for each TDP-43 variant in the absence and presence of PAR.All at room temperature.

    Techniques Used: Binding Assay, In Vitro, Sequencing, Variant Assay

    38) Product Images from "Defining the frequency of human papillomavirus and polyomavirus infection in urothelial bladder tumours"

    Article Title: Defining the frequency of human papillomavirus and polyomavirus infection in urothelial bladder tumours

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29438-y

    End-point PCR confirmation of polyomavirus qPCR results. The figure shows an agarose gel of PCR products generated using BKV or human GAPDH primers and DNA extracted from tumours. + indicates BKV positive tumours (C t s of 17.6, 22.5, 25.3 and 26.2 respectively, left to right) and − indicates BKV negative (C t > 35). The numbers on the left indicate the ladder sizes (bp). The predicted sizes for the 5 amplicons are 250, 281, 161, 164 and 269 bp respectively. The 5 PCRs were run separately (in parallel) and analysed on separate gels.
    Figure Legend Snippet: End-point PCR confirmation of polyomavirus qPCR results. The figure shows an agarose gel of PCR products generated using BKV or human GAPDH primers and DNA extracted from tumours. + indicates BKV positive tumours (C t s of 17.6, 22.5, 25.3 and 26.2 respectively, left to right) and − indicates BKV negative (C t > 35). The numbers on the left indicate the ladder sizes (bp). The predicted sizes for the 5 amplicons are 250, 281, 161, 164 and 269 bp respectively. The 5 PCRs were run separately (in parallel) and analysed on separate gels.

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Generated

    39) Product Images from "Loss of maternal ANNEXIN A10 via a 34-kb deleted-type copy number variation is associated with embryonic mortality in Japanese Black cattle"

    Article Title: Loss of maternal ANNEXIN A10 via a 34-kb deleted-type copy number variation is associated with embryonic mortality in Japanese Black cattle

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-3312-z

    CNVR_322 overlapping with ANXA10 gene region on chromosome 8. a Regional SNPs plot of CNVR_322. The mean log R ratio is indicated on the y -axis. The mean log R ratio of CNVR_322 animals ( magenta ) and the mean log R ratio of non-CNVR_322 animals ( blue ) were calculated from 35 animals. SNP positions were based on the UMD3.1 assembly of the bovine genome. b CNVR_322 ( black bar ) was visualized using the UCSC Genome Browser [ 54 ]. The Ensembl IDs and RefSeq gene symbol of ANXA10 were labeled. c qPCR validation of CNVR_322. The left-most bar represents a calibrator animal (JB_001). The calibrator animal is assumed to contain two copies of the DNA segment detected from the PennCNV analysis. Basic transcription factor 3 gene ( BTF3 ), which served as an internal qPCR standard for both copies at a locus (2n), was co-amplified with the primers. The x -axis represents the animals. Brackets represent the CNVR_322-detected animals using the Illumina BovineHD BeadChip Array. Error bars represent the ± Standard Error of the Mean obtained from three experiments
    Figure Legend Snippet: CNVR_322 overlapping with ANXA10 gene region on chromosome 8. a Regional SNPs plot of CNVR_322. The mean log R ratio is indicated on the y -axis. The mean log R ratio of CNVR_322 animals ( magenta ) and the mean log R ratio of non-CNVR_322 animals ( blue ) were calculated from 35 animals. SNP positions were based on the UMD3.1 assembly of the bovine genome. b CNVR_322 ( black bar ) was visualized using the UCSC Genome Browser [ 54 ]. The Ensembl IDs and RefSeq gene symbol of ANXA10 were labeled. c qPCR validation of CNVR_322. The left-most bar represents a calibrator animal (JB_001). The calibrator animal is assumed to contain two copies of the DNA segment detected from the PennCNV analysis. Basic transcription factor 3 gene ( BTF3 ), which served as an internal qPCR standard for both copies at a locus (2n), was co-amplified with the primers. The x -axis represents the animals. Brackets represent the CNVR_322-detected animals using the Illumina BovineHD BeadChip Array. Error bars represent the ± Standard Error of the Mean obtained from three experiments

    Techniques Used: Labeling, Real-time Polymerase Chain Reaction, Amplification

    40) Product Images from "ADAR promotes lung adenocarcinoma migration and invasion through stabilization of FAK"

    Article Title: ADAR promotes lung adenocarcinoma migration and invasion through stabilization of FAK

    Journal: Science signaling

    doi: 10.1126/scisignal.aah3941

    ADAR is overexpressed in lung adenocarcinoma (LUAD) and correlates with tumor recurrence (A) ADAR DNA copy numbers were determined by droplet digital PCR in human bronchial epithelial cells (HBECs) and the indicated LUAD cells. Data are in triplicate from three experiments. (B) ADAR mRNA expression in HBEC and the indicated LUAD cells were assessed by qRT-PCR. HPRT was amplified as a reference. Data are means ± SEM and in triplicate from three experiments. (C) Western blot of ADAR protein expression in HBEC and LUAD cells. N = 3 experiments. (D) Kaplan-Meier curve of progression-free survival based on ADAR mRNA expression in 162 stage I LUAD patients in the NCCRI cohort (log-rank test: p
    Figure Legend Snippet: ADAR is overexpressed in lung adenocarcinoma (LUAD) and correlates with tumor recurrence (A) ADAR DNA copy numbers were determined by droplet digital PCR in human bronchial epithelial cells (HBECs) and the indicated LUAD cells. Data are in triplicate from three experiments. (B) ADAR mRNA expression in HBEC and the indicated LUAD cells were assessed by qRT-PCR. HPRT was amplified as a reference. Data are means ± SEM and in triplicate from three experiments. (C) Western blot of ADAR protein expression in HBEC and LUAD cells. N = 3 experiments. (D) Kaplan-Meier curve of progression-free survival based on ADAR mRNA expression in 162 stage I LUAD patients in the NCCRI cohort (log-rank test: p

    Techniques Used: Digital PCR, Expressing, Quantitative RT-PCR, Amplification, Western Blot

    41) Product Images from "Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks"

    Article Title: Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn094

    Efficiency and fidelity of NHEJ depending on XRCC4. (A) Endjoining frequency after induction of DSBs by I-SceI endonuclease as measured by the frequency of XHATM- resistant colonies normalized to colony formation in non-selective medium. (B) Examples of repaired DNA sequences obtained from genomic DNA of individual XHATM resistant clones. Only the sense strand is shown. I-SceI recognition sites are depicted in bold. In the parental sequence, both start codons are underscored. Microhomologies are underscored within the example sequences. Fill-in synthesis is drawn as lower case letters. N indicates the total number of analyzed sequences. (C) Distribution of length of deletions at individual junctions. Deletions are defined as the sum of base pairs lost at both sites of the DSB. According to this definition, the 34-bp pop-out event in case of double I-SceI cleavage is not considered a deletion. (D) Distribution of the number of homologous bases (microhomologies) used for junction formation. Only terminal microhomologies are considered.
    Figure Legend Snippet: Efficiency and fidelity of NHEJ depending on XRCC4. (A) Endjoining frequency after induction of DSBs by I-SceI endonuclease as measured by the frequency of XHATM- resistant colonies normalized to colony formation in non-selective medium. (B) Examples of repaired DNA sequences obtained from genomic DNA of individual XHATM resistant clones. Only the sense strand is shown. I-SceI recognition sites are depicted in bold. In the parental sequence, both start codons are underscored. Microhomologies are underscored within the example sequences. Fill-in synthesis is drawn as lower case letters. N indicates the total number of analyzed sequences. (C) Distribution of length of deletions at individual junctions. Deletions are defined as the sum of base pairs lost at both sites of the DSB. According to this definition, the 34-bp pop-out event in case of double I-SceI cleavage is not considered a deletion. (D) Distribution of the number of homologous bases (microhomologies) used for junction formation. Only terminal microhomologies are considered.

    Techniques Used: Non-Homologous End Joining, Clone Assay, Sequencing

    Efficiency and fidelity of NHEJ depending on Ku80. (A) End-joining frequency after induction of DSBs by I-SceI endonuclease analogous to Figure 2A. (B) Examples of repaired DNA sequences obtained from genomic DNA of individual XHATM resistant clones. Display as in Figure 2, italic font indicates inserted nucleotides. (C) and (D) Distribution of deletion length and microhomology usage as in Figure 2.
    Figure Legend Snippet: Efficiency and fidelity of NHEJ depending on Ku80. (A) End-joining frequency after induction of DSBs by I-SceI endonuclease analogous to Figure 2A. (B) Examples of repaired DNA sequences obtained from genomic DNA of individual XHATM resistant clones. Display as in Figure 2, italic font indicates inserted nucleotides. (C) and (D) Distribution of deletion length and microhomology usage as in Figure 2.

    Techniques Used: Non-Homologous End Joining, Clone Assay

    42) Product Images from "DNA single-strand break-induced DNA damage response causes heart failure"

    Article Title: DNA single-strand break-induced DNA damage response causes heart failure

    Journal: Nature Communications

    doi: 10.1038/ncomms15104

    Accumulation of DNA SSB in the failing heart. ( a , b ) Cardiomyocytes were isolated from the TAC-operated heart at the indicated time points. The type of DNA damage in cardiomyocytes was assessed by comet assay. Representative images ( a ) and quantitative analyses are shown ( b , Alkaline comet: n =28, 45, 48; Neutral comet: n =38, 56, 44 at each time point, respectively, biological replicates=3). ( c , d ) Fragmented DNA and DSB were labelled with ISOL staining ( c , green). Wheat germ agglutinin (WGA, red) was used to visualize cardiomyocytes. DNase-treated section (DNase I, 10 Kunitz units ml −1 ) was used as a positive control. Arrowheads indicate ISOL-positive cardiomyocytes and arrows indicate ISOL-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. The number of ISOL-positive cardiomyocytes was counted ( d , n =4 each). ( e , f ) Heart tissue sections were immunostained for NBS1 ( e , NBS1, green). Immunostaining for alpha-actinin (red) was used to label cardiomyocytes. Scale bar, 50 μm. The number of NBS1-positive cardiomyocytes was counted ( f , n =4 each). ( g , h ) Heart tissue sections were immunostained for poly-ADP ribose ( g , PAR, green) and the number of PAR-positive cardiomyocytes was counted ( h , n =4, 4, 5 at each time point, respectively). Arrowheads indicate PAR-positive cardiomyocytes and arrows indicate PAR-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. ( i ) Expression levels of SSB repair enzymes were analysed by real-time PCR ( n =4, 6, 8 at each time point, respectively, technical duplicates). ( j , k ) Heart tissue sections were stained with dihydroethidium ( i , DHE, 10 μΜ) and mean fluorescence intensity relative to Sham-operated mice was measured ( k , n =4, 5, 5 at each time point, respectively). Scale bar, 50 μm. ( l ) The level of H 2 O 2 in the TAC-operated heart was measured using Amplex Red assay ( n =9, 5, 6 at each time point, respectively). Statistical significance was determined by Steel-Dwass test for ( b ) and by one-way analysis of variance followed by the Tukey–Kramer HSD test for ( d , f , h , i , j ) * P
    Figure Legend Snippet: Accumulation of DNA SSB in the failing heart. ( a , b ) Cardiomyocytes were isolated from the TAC-operated heart at the indicated time points. The type of DNA damage in cardiomyocytes was assessed by comet assay. Representative images ( a ) and quantitative analyses are shown ( b , Alkaline comet: n =28, 45, 48; Neutral comet: n =38, 56, 44 at each time point, respectively, biological replicates=3). ( c , d ) Fragmented DNA and DSB were labelled with ISOL staining ( c , green). Wheat germ agglutinin (WGA, red) was used to visualize cardiomyocytes. DNase-treated section (DNase I, 10 Kunitz units ml −1 ) was used as a positive control. Arrowheads indicate ISOL-positive cardiomyocytes and arrows indicate ISOL-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. The number of ISOL-positive cardiomyocytes was counted ( d , n =4 each). ( e , f ) Heart tissue sections were immunostained for NBS1 ( e , NBS1, green). Immunostaining for alpha-actinin (red) was used to label cardiomyocytes. Scale bar, 50 μm. The number of NBS1-positive cardiomyocytes was counted ( f , n =4 each). ( g , h ) Heart tissue sections were immunostained for poly-ADP ribose ( g , PAR, green) and the number of PAR-positive cardiomyocytes was counted ( h , n =4, 4, 5 at each time point, respectively). Arrowheads indicate PAR-positive cardiomyocytes and arrows indicate PAR-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. ( i ) Expression levels of SSB repair enzymes were analysed by real-time PCR ( n =4, 6, 8 at each time point, respectively, technical duplicates). ( j , k ) Heart tissue sections were stained with dihydroethidium ( i , DHE, 10 μΜ) and mean fluorescence intensity relative to Sham-operated mice was measured ( k , n =4, 5, 5 at each time point, respectively). Scale bar, 50 μm. ( l ) The level of H 2 O 2 in the TAC-operated heart was measured using Amplex Red assay ( n =9, 5, 6 at each time point, respectively). Statistical significance was determined by Steel-Dwass test for ( b ) and by one-way analysis of variance followed by the Tukey–Kramer HSD test for ( d , f , h , i , j ) * P

    Techniques Used: Isolation, Single Cell Gel Electrophoresis, Staining, Whole Genome Amplification, Positive Control, Immunostaining, Expressing, Real-time Polymerase Chain Reaction, Fluorescence, Mouse Assay, Amplex Red Assay

    43) Product Images from "Dynamic Interactions between Cancer Cells and the Embryonic Microenvironment Regulate Cell Invasion and Reveal EphB6 as a Metastasis Suppressor"

    Article Title: Dynamic Interactions between Cancer Cells and the Embryonic Microenvironment Regulate Cell Invasion and Reveal EphB6 as a Metastasis Suppressor

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-13-0673

    Re-expression of EphB6 in C8161 melanoma cells causes a significant loss of metastatic potential but does not affect tumorigenicity in a chorioallantoic membrane (CAM) metastasis assay A) Images of a native chick embryo CAM and representative tumors from parental C8161 cells (green) and EphB6+ C8161 cells (red) grown on the CAM. 1e6 cells in suspension (10ul volume) were dropped onto the CAM at day E10. Tumor formation occurred over 48 hours. Non-metastatic C81–61 cells did not form tumors. The black meter bar represents 1cm and the white meter bars represent 2.5mm. B) A cartoon depicting the CAM metastasis assay. Tumor cells are placed onto the upper CAM through a window in the egg shell. The egg is resealed for 48 hours. The lower CAM is removed and genomic DNA is harvested from the tissue. Metastatic human cells are detected by qPCR using primers that amplify a human-specific Alu element. C) A scatterplot showing detected amounts of human DNA per 2ug chick CAM DNA. Each X represents one biological replicate. A t-test was used to calculate statistical significance.
    Figure Legend Snippet: Re-expression of EphB6 in C8161 melanoma cells causes a significant loss of metastatic potential but does not affect tumorigenicity in a chorioallantoic membrane (CAM) metastasis assay A) Images of a native chick embryo CAM and representative tumors from parental C8161 cells (green) and EphB6+ C8161 cells (red) grown on the CAM. 1e6 cells in suspension (10ul volume) were dropped onto the CAM at day E10. Tumor formation occurred over 48 hours. Non-metastatic C81–61 cells did not form tumors. The black meter bar represents 1cm and the white meter bars represent 2.5mm. B) A cartoon depicting the CAM metastasis assay. Tumor cells are placed onto the upper CAM through a window in the egg shell. The egg is resealed for 48 hours. The lower CAM is removed and genomic DNA is harvested from the tissue. Metastatic human cells are detected by qPCR using primers that amplify a human-specific Alu element. C) A scatterplot showing detected amounts of human DNA per 2ug chick CAM DNA. Each X represents one biological replicate. A t-test was used to calculate statistical significance.

    Techniques Used: Expressing, Chick Chorioallantoic Membrane Assay, Real-time Polymerase Chain Reaction

    44) Product Images from "The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production"

    Article Title: The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production

    Journal: Scientific Reports

    doi: 10.1038/srep10152

    Identification of F0 transgenic cloned pigs. A) Detection of the rtTA gene in genomic DNA from blood samples of the 5 cloned pigs (lanes 4–8) and a non-transgenic pig (lane 2). Lane PC, using a pTTGH DNA fragment as a template; lane NC, using genomic DNA from non-transgenic pigs as a template. B) Transgenic pigs were identified using Southern blot and a Dig-labeled GH probe was used to hybridize the genomic DNA from ear tip tissue of transgenic pigs. After digestion with Eco RI, the endogenous GH (pGH1 in B) band and part of the pTTGH fragment (pGH2 in B) were detected in transgenic pigs. rtTA was also detected. The blots have been cropped to focus on the bands of interest. See Supplementary Fig. S4-5 for full-length gels.
    Figure Legend Snippet: Identification of F0 transgenic cloned pigs. A) Detection of the rtTA gene in genomic DNA from blood samples of the 5 cloned pigs (lanes 4–8) and a non-transgenic pig (lane 2). Lane PC, using a pTTGH DNA fragment as a template; lane NC, using genomic DNA from non-transgenic pigs as a template. B) Transgenic pigs were identified using Southern blot and a Dig-labeled GH probe was used to hybridize the genomic DNA from ear tip tissue of transgenic pigs. After digestion with Eco RI, the endogenous GH (pGH1 in B) band and part of the pTTGH fragment (pGH2 in B) were detected in transgenic pigs. rtTA was also detected. The blots have been cropped to focus on the bands of interest. See Supplementary Fig. S4-5 for full-length gels.

    Techniques Used: Transgenic Assay, Clone Assay, Southern Blot, Labeling

    45) Product Images from "A toolkit for rapid gene mapping in the nematode Caenorhabditis briggsae"

    Article Title: A toolkit for rapid gene mapping in the nematode Caenorhabditis briggsae

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-236

    Sub-chromosomal localization of mutations by medium and small indel-based mapping . The gel images show non-mutant (wild type, W) and mutant (M) pool of PCR amplified DNA from F2 worms. The histograms show ULVs for various indels. (A) dpy(sy5001) is most strongly linked to the medium indel cb-m197 (located roughly in the middle of chromosome X, ChrX-M) compared to flanking indels cb-m204 (left arm, ChrX-L) and cb-m136 (right arm, ChrX-R). (B) dpy(s1272), unc(sa972) , and lin(bh20) are located on chromosome 3. While dpy(s1272) and unc(sa972) are strongly linked to bhP14 and bhP18 and appear to be on the left arm, lin(bh20) maps closer to bhP40 (center right region). (C) unc(sy5422) is tightly linked to indels bhP9 and bhP16 on the right arm of chromosome 4.
    Figure Legend Snippet: Sub-chromosomal localization of mutations by medium and small indel-based mapping . The gel images show non-mutant (wild type, W) and mutant (M) pool of PCR amplified DNA from F2 worms. The histograms show ULVs for various indels. (A) dpy(sy5001) is most strongly linked to the medium indel cb-m197 (located roughly in the middle of chromosome X, ChrX-M) compared to flanking indels cb-m204 (left arm, ChrX-L) and cb-m136 (right arm, ChrX-R). (B) dpy(s1272), unc(sa972) , and lin(bh20) are located on chromosome 3. While dpy(s1272) and unc(sa972) are strongly linked to bhP14 and bhP18 and appear to be on the left arm, lin(bh20) maps closer to bhP40 (center right region). (C) unc(sy5422) is tightly linked to indels bhP9 and bhP16 on the right arm of chromosome 4.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification

    Mutation mapping by polymorphisms . The indels were used to map dpy(sy5001) (A) and dpy(sy5148) (B, C) and snip-SNP (cb650) was used to map lin(bh25) (D). (A) Mapping of X-linked mutation dpy(sy5001) using six medium indels (one per chromosome). W, non-mutant (phenotypically wild type) pool; M, mutant pool. (B) dpy(sy5148) localization on chromosome 2 by small indels (Chr 1: bhP19, Chr 2: bhP21, Chr 3: bhP12, Chr 4: bhP11 and Chr X: bhP26). (C) ULVs for dpy(sy5148) show linkage to bhP21. X-axis shows chromosomes whereas Y-axis linkage values. The dotted line shows the baseline for unlinked chromosomes. (D) Sac I digested PCR amplified genomic DNA of wild-type controls (A: AF16, H: HK104) and lin(bh25) mutant (M) and non-mutant (W) categories. There is a clear bias towards AF16 DNA (uncut) in the mutant pool compared to the non-mutant pool, demonstrating that lin(bh25) is linked to cb650 (chr. 1).
    Figure Legend Snippet: Mutation mapping by polymorphisms . The indels were used to map dpy(sy5001) (A) and dpy(sy5148) (B, C) and snip-SNP (cb650) was used to map lin(bh25) (D). (A) Mapping of X-linked mutation dpy(sy5001) using six medium indels (one per chromosome). W, non-mutant (phenotypically wild type) pool; M, mutant pool. (B) dpy(sy5148) localization on chromosome 2 by small indels (Chr 1: bhP19, Chr 2: bhP21, Chr 3: bhP12, Chr 4: bhP11 and Chr X: bhP26). (C) ULVs for dpy(sy5148) show linkage to bhP21. X-axis shows chromosomes whereas Y-axis linkage values. The dotted line shows the baseline for unlinked chromosomes. (D) Sac I digested PCR amplified genomic DNA of wild-type controls (A: AF16, H: HK104) and lin(bh25) mutant (M) and non-mutant (W) categories. There is a clear bias towards AF16 DNA (uncut) in the mutant pool compared to the non-mutant pool, demonstrating that lin(bh25) is linked to cb650 (chr. 1).

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification

    46) Product Images from "Mutations in ANKRD11 Cause KBG Syndrome, Characterized by Intellectual Disability, Skeletal Malformations, and Macrodontia"

    Article Title: Mutations in ANKRD11 Cause KBG Syndrome, Characterized by Intellectual Disability, Skeletal Malformations, and Macrodontia

    Journal: American Journal of Human Genetics

    doi: 10.1016/j.ajhg.2011.06.007

    Activity-Dependent Nuclear Aggregation of ANKRD11 (A) RT-PCR expression analysis of ANKRD11 in adult human brain cDNA. (1) DNA control sample, (2) cDNA sample with reverse transcriptase, and (3) cDNA sample without reverse transcriptase in RT reaction. (B) Representative image of neonatal cortical neurons transfected with GFP-ANKRD11 along with DsRed-MECP2. Postnatal day 1 neurons (cultured in vitro for 5 days) were cotransfected with GFP-ANKRD11 and Ds-Red-MECP2. Twenty-four hours after transfection, 50 mM KCl (or 5mM to controls plates) was added to the culture media for 90 min, cells were fixed, stained with antibodies to tyrosinated tubulin (blue), and imaged on a confocal microscope. (C) Higher magnification image of cotransfected neurons to observe GFP-ANKRD11 nuclear inclusions (arrows). GFP-ANKRD11 (green) and DsRed-MeCP2 (red) nuclear aggregates do not colocalize. (D) Representative glial cells (identified by their lack of tyrosinated-tubulin staining) from the same cultures as in (B). Note that in glial cells, ANKRD11 nuclear inclusions form regardless of KCl addition.
    Figure Legend Snippet: Activity-Dependent Nuclear Aggregation of ANKRD11 (A) RT-PCR expression analysis of ANKRD11 in adult human brain cDNA. (1) DNA control sample, (2) cDNA sample with reverse transcriptase, and (3) cDNA sample without reverse transcriptase in RT reaction. (B) Representative image of neonatal cortical neurons transfected with GFP-ANKRD11 along with DsRed-MECP2. Postnatal day 1 neurons (cultured in vitro for 5 days) were cotransfected with GFP-ANKRD11 and Ds-Red-MECP2. Twenty-four hours after transfection, 50 mM KCl (or 5mM to controls plates) was added to the culture media for 90 min, cells were fixed, stained with antibodies to tyrosinated tubulin (blue), and imaged on a confocal microscope. (C) Higher magnification image of cotransfected neurons to observe GFP-ANKRD11 nuclear inclusions (arrows). GFP-ANKRD11 (green) and DsRed-MeCP2 (red) nuclear aggregates do not colocalize. (D) Representative glial cells (identified by their lack of tyrosinated-tubulin staining) from the same cultures as in (B). Note that in glial cells, ANKRD11 nuclear inclusions form regardless of KCl addition.

    Techniques Used: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Cell Culture, In Vitro, Staining, Microscopy

    47) Product Images from "REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice"

    Article Title: REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07060-w

    Rex1 knockout mice are viable and fertile. a BAC targeting strategy to generate the Rex1 knockout cas allele in 129:cas ESCs. The Rex1 coding sequence is shown as a blue box. The start site is indicated by a black arrow. LoxP sites, denoted as red triangles, flank the neomycin-resistance gene (neo) as a positive gene selection marker. Primers used to validate gene recombination in ESCs and to genotype are shown as red arrows. b Validation of Rex1 knockout cas allele recombination by XmnI RFLP analysis of WT and Rex1 -targeted ESCs (top panel). Pf1M1 RFLP analysis to detect presence of two X chromosomes (bottom panel). c Nuclear extracts of WT, Rex1 +/− and Rex1 −/− ESCs were immunoblotted with RNF12 and REX1 antibodies. ACTIN was used as a loading control. Uncropped WB images are found in Supplementary Fig. 10b . d Sex and genotype distribution from different matings of Rex1 -deficient mice. Number of breedings, number of mice per breeding and total number of mice are indicated below. No significant bias against the birth of female animals was observed ( χ 2 test, p > 0.05). e Xist RNA-FISH (FITC) analysis on WT, Rex1 +/− and Rex1 −/− ESCs at day 3 of differentiation. DNA was stained with DAPI (blue). White arrows indicate the presence of two clouds within a nucleus. Scale bar: 20 μm. f Quantification of Xist -positive cells (left panel) and Xist -positive cells with two clouds (right panel) in WT, Rex1 +/− and Rex1 −/− ESCs at day 0 and day 3 of differentiation. Asterisks indicate p -value
    Figure Legend Snippet: Rex1 knockout mice are viable and fertile. a BAC targeting strategy to generate the Rex1 knockout cas allele in 129:cas ESCs. The Rex1 coding sequence is shown as a blue box. The start site is indicated by a black arrow. LoxP sites, denoted as red triangles, flank the neomycin-resistance gene (neo) as a positive gene selection marker. Primers used to validate gene recombination in ESCs and to genotype are shown as red arrows. b Validation of Rex1 knockout cas allele recombination by XmnI RFLP analysis of WT and Rex1 -targeted ESCs (top panel). Pf1M1 RFLP analysis to detect presence of two X chromosomes (bottom panel). c Nuclear extracts of WT, Rex1 +/− and Rex1 −/− ESCs were immunoblotted with RNF12 and REX1 antibodies. ACTIN was used as a loading control. Uncropped WB images are found in Supplementary Fig. 10b . d Sex and genotype distribution from different matings of Rex1 -deficient mice. Number of breedings, number of mice per breeding and total number of mice are indicated below. No significant bias against the birth of female animals was observed ( χ 2 test, p > 0.05). e Xist RNA-FISH (FITC) analysis on WT, Rex1 +/− and Rex1 −/− ESCs at day 3 of differentiation. DNA was stained with DAPI (blue). White arrows indicate the presence of two clouds within a nucleus. Scale bar: 20 μm. f Quantification of Xist -positive cells (left panel) and Xist -positive cells with two clouds (right panel) in WT, Rex1 +/− and Rex1 −/− ESCs at day 0 and day 3 of differentiation. Asterisks indicate p -value

    Techniques Used: Knock-Out, Mouse Assay, BAC Assay, Sequencing, Selection, Marker, Western Blot, Fluorescence In Situ Hybridization, Staining

    48) Product Images from "Cyclin-dependent kinase 2 is an ideal target for ovary tumors with elevated cyclin E1 expression"

    Article Title: Cyclin-dependent kinase 2 is an ideal target for ovary tumors with elevated cyclin E1 expression

    Journal: Oncotarget

    doi:

    The status of CCNE1 overexpression is not associated with tumorigenic behaviors in established ovarian cancer cells A. Total RNA was isolated from overnight-cultured cells and subjected to qRT-PCR to quantitate the amount of CCNE1 mRNA. β-actin mRNA was used as an internal standard for normalization. B. Overnight-cultured cells were harvested and cell lysates were subjected to immunoblotting to detect CCNE1 protein using CCNE1 mAb. Membrane was stripped and reprobled with GAPDH polyclonal antibody for loading normalization. C. Genomic DNA was isolated from overnight-cultured cells and subjected to qPCR to analyze copy number of CCNE1 gene. ACTB gene was used for normalization. D. Cells (50,000/well) were plated in 24-well plates for overnight and then cultured for 2 days followed by MTT assay. % of growth increase was calculated as [(OD overnight –; OD end )/OD overnight ] x 100. E. Cells (1×10 7 cell/mouse) from various ovarian cancer lines were intraperitoneally injected to nude mice for 4 weeks to allow metastatic colonization. Images are the views of various areas in peritoneal cavity. Arrows point to metastatic implants. F. Metastatic implants were collected and weighed. Data are means ± SE. n = 6.
    Figure Legend Snippet: The status of CCNE1 overexpression is not associated with tumorigenic behaviors in established ovarian cancer cells A. Total RNA was isolated from overnight-cultured cells and subjected to qRT-PCR to quantitate the amount of CCNE1 mRNA. β-actin mRNA was used as an internal standard for normalization. B. Overnight-cultured cells were harvested and cell lysates were subjected to immunoblotting to detect CCNE1 protein using CCNE1 mAb. Membrane was stripped and reprobled with GAPDH polyclonal antibody for loading normalization. C. Genomic DNA was isolated from overnight-cultured cells and subjected to qPCR to analyze copy number of CCNE1 gene. ACTB gene was used for normalization. D. Cells (50,000/well) were plated in 24-well plates for overnight and then cultured for 2 days followed by MTT assay. % of growth increase was calculated as [(OD overnight –; OD end )/OD overnight ] x 100. E. Cells (1×10 7 cell/mouse) from various ovarian cancer lines were intraperitoneally injected to nude mice for 4 weeks to allow metastatic colonization. Images are the views of various areas in peritoneal cavity. Arrows point to metastatic implants. F. Metastatic implants were collected and weighed. Data are means ± SE. n = 6.

    Techniques Used: Over Expression, Isolation, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, MTT Assay, Injection, Mouse Assay

    49) Product Images from "Preconditioning lessens high fat induced metabolic syndrome along with markers of increased metabolic capacity in muscle and adipose tissue"

    Article Title: Preconditioning lessens high fat induced metabolic syndrome along with markers of increased metabolic capacity in muscle and adipose tissue

    Journal: Bioscience Reports

    doi: 10.1042/BSR20181873

    Effects of HF prior exposures in muscle Quadriceps muscle was collected from the mice at the end of the study (week 14) for the following measurements. ( A ) Protein levels of AMPK and mitochondrial complex measured by Western blotting and normalised by GAPDH; ( B ) β-HAD activity was measured by the disappearance rate of NADH, and CiS activity (CiS) was detected by detecting the rate of the CoA-SH production using DTNB. ( C ) Levels of mRNA expression of PGC1α and UCP3 and ( D ) nuclear respiratory factor-1 (NRF1) were quantified by qRT-PCR. ( E ) mtDNA content was measured by quantificative PCR using MCT-1 (genomic DNA marker) as a control gene. † P
    Figure Legend Snippet: Effects of HF prior exposures in muscle Quadriceps muscle was collected from the mice at the end of the study (week 14) for the following measurements. ( A ) Protein levels of AMPK and mitochondrial complex measured by Western blotting and normalised by GAPDH; ( B ) β-HAD activity was measured by the disappearance rate of NADH, and CiS activity (CiS) was detected by detecting the rate of the CoA-SH production using DTNB. ( C ) Levels of mRNA expression of PGC1α and UCP3 and ( D ) nuclear respiratory factor-1 (NRF1) were quantified by qRT-PCR. ( E ) mtDNA content was measured by quantificative PCR using MCT-1 (genomic DNA marker) as a control gene. † P

    Techniques Used: Mouse Assay, Western Blot, Activity Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Marker

    50) Product Images from "A Downy Mildew Effector Attenuates Salicylic Acid-Triggered Immunity in Arabidopsis by Interacting with the Host Mediator Complex"

    Article Title: A Downy Mildew Effector Attenuates Salicylic Acid-Triggered Immunity in Arabidopsis by Interacting with the Host Mediator Complex

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001732

    MED19a is a positive regulator of nuclear immunity against Hpa . (A) Schematic diagram of T-DNA insertions in MED19a . (B) MED19a expression in med19a-1 and med19a-2 mutants. (C) Representative images of the phenotype observed in 4-wk-old floral stem of Col-0, med19a-1 , med19a-2 , and med19a mutant complemented line C1. (D) Developmental phenotype of Arabidopsis transgenic lines OE-MED19a compared to Col-0. (E) Immunoblot of the Co-immunoprecipitation analysis between GFP-MED19a and MED6 and MED7. Arrows point out the interaction detected between GFP-MED19a and MED6 and MED7. (F) Subcellular localisation of GFP-MED19a in Arabidopsis plant. Scale bar, 5 µm. (G) Immunoblot of proteins extracted from two independent lines expressing GFP-MED19a. Stars indicate the expected size for GFP-MED19a. Notice the upper bands in the blot that might suggest posttranscriptional modifications. (H) Monitoring of Hpa sporulation at 5 DAI in control lines (Col-0 and GFP), med19a mutant complemented lines (C1 and C2), Mediator mutants, and MED19a OE lines. Error bars represent the standard error of the mean. Asterisks represent the significance of individual unpaired t tests comparing the given column with the control ( p value
    Figure Legend Snippet: MED19a is a positive regulator of nuclear immunity against Hpa . (A) Schematic diagram of T-DNA insertions in MED19a . (B) MED19a expression in med19a-1 and med19a-2 mutants. (C) Representative images of the phenotype observed in 4-wk-old floral stem of Col-0, med19a-1 , med19a-2 , and med19a mutant complemented line C1. (D) Developmental phenotype of Arabidopsis transgenic lines OE-MED19a compared to Col-0. (E) Immunoblot of the Co-immunoprecipitation analysis between GFP-MED19a and MED6 and MED7. Arrows point out the interaction detected between GFP-MED19a and MED6 and MED7. (F) Subcellular localisation of GFP-MED19a in Arabidopsis plant. Scale bar, 5 µm. (G) Immunoblot of proteins extracted from two independent lines expressing GFP-MED19a. Stars indicate the expected size for GFP-MED19a. Notice the upper bands in the blot that might suggest posttranscriptional modifications. (H) Monitoring of Hpa sporulation at 5 DAI in control lines (Col-0 and GFP), med19a mutant complemented lines (C1 and C2), Mediator mutants, and MED19a OE lines. Error bars represent the standard error of the mean. Asterisks represent the significance of individual unpaired t tests comparing the given column with the control ( p value

    Techniques Used: Expressing, Mutagenesis, Transgenic Assay, Immunoprecipitation

    51) Product Images from "Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood"

    Article Title: Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20111316

    Heterozygous TBK1 mutations in two children with HSE. (a) Family pedigrees and segregation. (b) Heterozygous TBK1 mutations 476G > C in P1 and 149A > C in P2. The PCR products sequenced were amplified from genomic DNA from the granulocytes of a control (C) and both patients. (c) Schematic diagram of the protein structure of TBK1, featuring its kinase domain (KD), ubiquitin-like domain (ULD), and coiled-coil (CC) regions. Both heterozygous substitutions, 159G > G/A (P1) and 50D > D/A (P2), affect the kinase domain of TBK1 (amino acids 9–305). (d) Multiple alignments of relevant amino acid sequences of the kinase domain of human TBK1 with its homologues from nine other species, with the residues mutated in P1 (G159) and P2 (D50) highlighted. (e) Multiple alignments of relevant amino acid sequences of the kinase domain of human TBK1 with the other IKK and IKK-related kinases, IKK-α (46% similar to TBK1), IKK-β (44% similar), and IKK-ε (64% similar). The residues mutated in P1 and P2 are conserved (G159) or similar (D50) across IKK or IKK-related kinases. (Blue signifies sequence similarity, teal signifies sequence identity, and gray signifies partial identity/similarity.) (f) TBK1 expression, as assessed by RT-qPCR on mRNA from the SV40-fibroblasts of patients (P1 and P2) and control lines (C+1 and C+2). Values represent mean values ± SD calculated from three independent experiments. (g) TBK1 levels, as assessed by Western blotting, in SV40-fibroblasts from patients (P1 and P2) and two control lines (C+ 1 and C+ 2). This Western blot result is representative of three experiments.
    Figure Legend Snippet: Heterozygous TBK1 mutations in two children with HSE. (a) Family pedigrees and segregation. (b) Heterozygous TBK1 mutations 476G > C in P1 and 149A > C in P2. The PCR products sequenced were amplified from genomic DNA from the granulocytes of a control (C) and both patients. (c) Schematic diagram of the protein structure of TBK1, featuring its kinase domain (KD), ubiquitin-like domain (ULD), and coiled-coil (CC) regions. Both heterozygous substitutions, 159G > G/A (P1) and 50D > D/A (P2), affect the kinase domain of TBK1 (amino acids 9–305). (d) Multiple alignments of relevant amino acid sequences of the kinase domain of human TBK1 with its homologues from nine other species, with the residues mutated in P1 (G159) and P2 (D50) highlighted. (e) Multiple alignments of relevant amino acid sequences of the kinase domain of human TBK1 with the other IKK and IKK-related kinases, IKK-α (46% similar to TBK1), IKK-β (44% similar), and IKK-ε (64% similar). The residues mutated in P1 and P2 are conserved (G159) or similar (D50) across IKK or IKK-related kinases. (Blue signifies sequence similarity, teal signifies sequence identity, and gray signifies partial identity/similarity.) (f) TBK1 expression, as assessed by RT-qPCR on mRNA from the SV40-fibroblasts of patients (P1 and P2) and control lines (C+1 and C+2). Values represent mean values ± SD calculated from three independent experiments. (g) TBK1 levels, as assessed by Western blotting, in SV40-fibroblasts from patients (P1 and P2) and two control lines (C+ 1 and C+ 2). This Western blot result is representative of three experiments.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Expressing, Quantitative RT-PCR, Western Blot

    52) Product Images from "Oxygen nanobubbles revert hypoxia by methylation programming"

    Article Title: Oxygen nanobubbles revert hypoxia by methylation programming

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08988-7

    Quantitative real-time PCR, LC-MS, and gene-specific methylation analysis of in vivo tumors. ( a ) LC - MS/MS quantitation of 5mC levels in terms of ratios of 5-methyl-2′-deoxycytidine (5mdC) to that of deoxycytidine (dC) in nanobubble shell (CTRL) and oxygen nanobubble (Treatment) treated groups for HeLa (left) and MB49 (right) cells in mice models. ( b ) Transcription levels of HIF-1α, PDK-1, and MAT2A determined by qRT-PCR. (n = 16, p-value was calculated by ANOVA). ( c ) Changes in promoter methylation of the 22 selected tumor suppressor genes. ( d ) Oxygen nanobubble induced changes in DNA methylation at the CpG islands of Human BRCA1 promoter region. The extent of methylation was compared between in vivo tumors treated with either nanobubbles or saline. Methylation levels were measured by bisulfite treatment of the DNA followed by pyrosequencing and expressed as % change at the promoter region of chromosome 17. *** P
    Figure Legend Snippet: Quantitative real-time PCR, LC-MS, and gene-specific methylation analysis of in vivo tumors. ( a ) LC - MS/MS quantitation of 5mC levels in terms of ratios of 5-methyl-2′-deoxycytidine (5mdC) to that of deoxycytidine (dC) in nanobubble shell (CTRL) and oxygen nanobubble (Treatment) treated groups for HeLa (left) and MB49 (right) cells in mice models. ( b ) Transcription levels of HIF-1α, PDK-1, and MAT2A determined by qRT-PCR. (n = 16, p-value was calculated by ANOVA). ( c ) Changes in promoter methylation of the 22 selected tumor suppressor genes. ( d ) Oxygen nanobubble induced changes in DNA methylation at the CpG islands of Human BRCA1 promoter region. The extent of methylation was compared between in vivo tumors treated with either nanobubbles or saline. Methylation levels were measured by bisulfite treatment of the DNA followed by pyrosequencing and expressed as % change at the promoter region of chromosome 17. *** P

    Techniques Used: Real-time Polymerase Chain Reaction, Liquid Chromatography with Mass Spectroscopy, Methylation, In Vivo, Mass Spectrometry, Quantitation Assay, Mouse Assay, Quantitative RT-PCR, DNA Methylation Assay

    53) Product Images from "A single clonal lineage of transmissible cancer identified in two marine mussel species in South America and Europe"

    Article Title: A single clonal lineage of transmissible cancer identified in two marine mussel species in South America and Europe

    Journal: eLife

    doi: 10.7554/eLife.47788

    Phylogenetic analysis of mitochondrial CR alleles from normal and diseased mussels. The CR region of mitochondrial DNA was amplified, and multiple alleles were cloned from different individual normal and diseased mussels of different species and locations: M. trossulus from BC (black), M. chilensis from Argentina and Chile (red), and M. edulis from France and the Netherlands (blue). ( A ) A list of cloned alleles is shown, with filled boxes marking cancer-associated alleles. ( B ) A schematic of the rearrangement found in the cancer-associated alleles is shown with black boxes representing female-derived sequence and white boxes representing male-derived sequence (gray bars represent the likely recombination region). Parentheses mark the repeat region in allele D. ( C ) Phylogenetic analysis of aligned alleles shows groups of related alleles (see Figure 3—source data 1 ). Names specify individual ID and allele ID. Alleles from normal individuals and host alleles from diseased individuals are marked with open circles (female allele) and open squares (male allele). Closed circles mark cancer-associated alleles (colored by host species). The tree was rooted at the midpoint, with bootstrap values below 50 removed. Model used was GTR+I. The scale bar marks genetic distance. Multiple male alleles were identified in M. chilensis samples, and a representative with the greatest number of clones was chosen for each individual. Reference sequences include female M. trossulus (AY823625.1), male M. trossulus (HM462081.1), female M. edulis (DQ198231.2), male M. edulis (AY823623.1), and the M. trossulus recombinant 62cm10 (KM192133.1). 10.7554/eLife.47788.008 FASTA formatted text file of mtCR sequence alignment.
    Figure Legend Snippet: Phylogenetic analysis of mitochondrial CR alleles from normal and diseased mussels. The CR region of mitochondrial DNA was amplified, and multiple alleles were cloned from different individual normal and diseased mussels of different species and locations: M. trossulus from BC (black), M. chilensis from Argentina and Chile (red), and M. edulis from France and the Netherlands (blue). ( A ) A list of cloned alleles is shown, with filled boxes marking cancer-associated alleles. ( B ) A schematic of the rearrangement found in the cancer-associated alleles is shown with black boxes representing female-derived sequence and white boxes representing male-derived sequence (gray bars represent the likely recombination region). Parentheses mark the repeat region in allele D. ( C ) Phylogenetic analysis of aligned alleles shows groups of related alleles (see Figure 3—source data 1 ). Names specify individual ID and allele ID. Alleles from normal individuals and host alleles from diseased individuals are marked with open circles (female allele) and open squares (male allele). Closed circles mark cancer-associated alleles (colored by host species). The tree was rooted at the midpoint, with bootstrap values below 50 removed. Model used was GTR+I. The scale bar marks genetic distance. Multiple male alleles were identified in M. chilensis samples, and a representative with the greatest number of clones was chosen for each individual. Reference sequences include female M. trossulus (AY823625.1), male M. trossulus (HM462081.1), female M. edulis (DQ198231.2), male M. edulis (AY823623.1), and the M. trossulus recombinant 62cm10 (KM192133.1). 10.7554/eLife.47788.008 FASTA formatted text file of mtCR sequence alignment.

    Techniques Used: Amplification, Clone Assay, Derivative Assay, Sequencing, Recombinant

    54) Product Images from "epiG: statistical inference and profiling of DNA methylation from whole-genome bisulfite sequencing data"

    Article Title: epiG: statistical inference and profiling of DNA methylation from whole-genome bisulfite sequencing data

    Journal: Genome Biology

    doi: 10.1186/s13059-017-1168-4

    DNA accessibility near a CTCF site. a Consensus DNA accessibility near a representative CTCF site at position 31,109,691 on chromosome 20 in the LNCaP sample. The center of the CTCF site is at position 0 of the plot. Red line : consensus based on the two chains shown in ( b ); blue line : consensus based on all reads. b The two dominating epi-alleles. Each point is an isolated GpC position. Black : methylated GpC position (DNA accessible), white : unmethylated GpC position (DNA inaccessible). The approximate lengths of the inaccessible intervals are computed ( black lines ), showing that the CTCF site appears to be flanked by nucleosomes (∼149 bp). In one epi-allele, the CTCF site appears to be inaccessible, potentially due to binding of a protein. As nucleosome positioning is dynamic and the read coverage is low for these samples, the inferred intervals are uncertain
    Figure Legend Snippet: DNA accessibility near a CTCF site. a Consensus DNA accessibility near a representative CTCF site at position 31,109,691 on chromosome 20 in the LNCaP sample. The center of the CTCF site is at position 0 of the plot. Red line : consensus based on the two chains shown in ( b ); blue line : consensus based on all reads. b The two dominating epi-alleles. Each point is an isolated GpC position. Black : methylated GpC position (DNA accessible), white : unmethylated GpC position (DNA inaccessible). The approximate lengths of the inaccessible intervals are computed ( black lines ), showing that the CTCF site appears to be flanked by nucleosomes (∼149 bp). In one epi-allele, the CTCF site appears to be inaccessible, potentially due to binding of a protein. As nucleosome positioning is dynamic and the read coverage is low for these samples, the inferred intervals are uncertain

    Techniques Used: Isolation, Gel Permeation Chromatography, Methylation, Binding Assay

    55) Product Images from "Progressive GAA?TTC Repeat Expansion in Human Cell Lines"

    Article Title: Progressive GAA?TTC Repeat Expansion in Human Cell Lines

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1000704

    Model of GAA·TTC repeat expansion in human cell lines. (A) Tandem reporter construct designed to isolate transcription elongation through GAA·TTC repeat insert sequences. Single copy integration of the construct into the genome of the host cell line is facilitated via conservative, site-specific recombination using Flp-recombinase. (B) Southern blot analysis of GAA·TTC repeat expansion using the template DNA isolated from a human cell line with a (GAA·TTC) 352 insert after 0, 1, 2, 4, and 10 weeks (W0–W10) in culture. M: 1 Kb plus size standard. EcoRV digestion of the genomic DNA cuts the tandem construct upstream of the GAA·TTC repeat region and between the 5′ hRLUC and 3′ hRLUC regions. 5′ hRLUC probe is specific to the 5′ side of the hRLUC reporter in the tandem construct containing the GAA·TTC insert region. 3′ hRLUC probe is specific to the 3′ region of the hRLUC expression cassette in the tandem construct and is not associated with the GAA·TTC repeat region. (C) PCR analysis of a (GAA·TTC) 352 repeat insert isolated at W0, W1, W2, W4, and W10 from Figure 1B . PCR amplification adds 438 bp to the GAA·TTC insert (5′: 338 bp+(GAA) n +100 bp: 3′). M: 1 Kb plus size standard (D) PCR analysis of GAA·TTC repeat inserts from clonal cell isolates derived from an end-point dilution of the (GAA·TTC) 352 parental cell line at W4 in Figure 1B and 1C . PCR amplification adds 438 bp to the GAA·TTC insert. M: 1 Kb plus size standard.
    Figure Legend Snippet: Model of GAA·TTC repeat expansion in human cell lines. (A) Tandem reporter construct designed to isolate transcription elongation through GAA·TTC repeat insert sequences. Single copy integration of the construct into the genome of the host cell line is facilitated via conservative, site-specific recombination using Flp-recombinase. (B) Southern blot analysis of GAA·TTC repeat expansion using the template DNA isolated from a human cell line with a (GAA·TTC) 352 insert after 0, 1, 2, 4, and 10 weeks (W0–W10) in culture. M: 1 Kb plus size standard. EcoRV digestion of the genomic DNA cuts the tandem construct upstream of the GAA·TTC repeat region and between the 5′ hRLUC and 3′ hRLUC regions. 5′ hRLUC probe is specific to the 5′ side of the hRLUC reporter in the tandem construct containing the GAA·TTC insert region. 3′ hRLUC probe is specific to the 3′ region of the hRLUC expression cassette in the tandem construct and is not associated with the GAA·TTC repeat region. (C) PCR analysis of a (GAA·TTC) 352 repeat insert isolated at W0, W1, W2, W4, and W10 from Figure 1B . PCR amplification adds 438 bp to the GAA·TTC insert (5′: 338 bp+(GAA) n +100 bp: 3′). M: 1 Kb plus size standard (D) PCR analysis of GAA·TTC repeat inserts from clonal cell isolates derived from an end-point dilution of the (GAA·TTC) 352 parental cell line at W4 in Figure 1B and 1C . PCR amplification adds 438 bp to the GAA·TTC insert. M: 1 Kb plus size standard.

    Techniques Used: Construct, Southern Blot, Isolation, Expressing, Polymerase Chain Reaction, Amplification, Derivative Assay

    56) Product Images from "Human endogenous retroviral K element encodes fusogenic activity in melanoma cells"

    Article Title: Human endogenous retroviral K element encodes fusogenic activity in melanoma cells

    Journal: Journal of Carcinogenesis

    doi: 10.4103/1477-3163.109032

    Detection of genetic changes in post-cell fusion cells and a proposed model of cell fusion in tumor progression (a). Ten single cell clones including 2 pS-puro-scrambled (clones 1-2), 2 pEYFP-N3-neo (clones 3-4), and 6 pS-puro-scrambled-pEYFP-N3-neo double selected clones (clones 5-10) were selected at random and amplified in cell culture. Genomic DNA was extracted from the cells. AmpFlSTR Identifiler PCR Amplification kit was used to examine microsatellite markers. Shown are fragment analysis of the D19S433 alleles. Note the fragment height of allele a (in the red box) was decreased in the fusion cells (6-9), and lost completely in clone #10 (b). A proposed model of K type human endogenous retrovirus in mediating intercellular fusion, evolution of genetic changes, and malignant progression
    Figure Legend Snippet: Detection of genetic changes in post-cell fusion cells and a proposed model of cell fusion in tumor progression (a). Ten single cell clones including 2 pS-puro-scrambled (clones 1-2), 2 pEYFP-N3-neo (clones 3-4), and 6 pS-puro-scrambled-pEYFP-N3-neo double selected clones (clones 5-10) were selected at random and amplified in cell culture. Genomic DNA was extracted from the cells. AmpFlSTR Identifiler PCR Amplification kit was used to examine microsatellite markers. Shown are fragment analysis of the D19S433 alleles. Note the fragment height of allele a (in the red box) was decreased in the fusion cells (6-9), and lost completely in clone #10 (b). A proposed model of K type human endogenous retrovirus in mediating intercellular fusion, evolution of genetic changes, and malignant progression

    Techniques Used: Clone Assay, Amplification, Cell Culture, Polymerase Chain Reaction

    57) Product Images from "Phenotypic and Molecular Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Bangladesh"

    Article Title: Phenotypic and Molecular Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Bangladesh

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108735

    PFGE banding patterns of Xba I-digested chromosomal DNA of representative ESBL-producing E. coli isolates. Lane 1, Salmonella enterica serovar Braenderup (H9812) (marker); Lanes 2-5, PFGE type A; Lane 6, PFGE type B; Lane 7, PFGE type P; Lane 8, Salmonella enterica serovar Braenderup (H9812) (marker); Lane 9, PFGE type E; Lane 10, PFGE type C; Lane 11, PFGE type F; Lane 12, PFGE type G; Lane 13, PFGE type B. Four isolates belonged to PFGE pattern A were isolated from UTI patients attending SMCH hospital.
    Figure Legend Snippet: PFGE banding patterns of Xba I-digested chromosomal DNA of representative ESBL-producing E. coli isolates. Lane 1, Salmonella enterica serovar Braenderup (H9812) (marker); Lanes 2-5, PFGE type A; Lane 6, PFGE type B; Lane 7, PFGE type P; Lane 8, Salmonella enterica serovar Braenderup (H9812) (marker); Lane 9, PFGE type E; Lane 10, PFGE type C; Lane 11, PFGE type F; Lane 12, PFGE type G; Lane 13, PFGE type B. Four isolates belonged to PFGE pattern A were isolated from UTI patients attending SMCH hospital.

    Techniques Used: Marker, Isolation

    58) Product Images from "Homeobox oncogene activation by pan-cancer DNA hypermethylation"

    Article Title: Homeobox oncogene activation by pan-cancer DNA hypermethylation

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1492-3

    Gene-body canyon hypermethylation by dCas9-SunTag-DNMT3A can directly increase oncogene DLX1 expression. a Schematic graph for dCas9-SunTag-DNMT3A DNA methylation editing system. De-activated Cas9 (dCas9) was fused to SunTag epitopes and single-chain variable fragment (scFv) was fused to GFP and DNMT3A to methylate the gene-body canyon and activate gene expression. b Genome browser tracks of gene-body hypermethylated canyons around homeobox gene DLX1 across 30 normal and 35 tumor samples. c CpG DNA methylation level dramatically increased at the gene-body of DLX1 after adding guide RNA DLX1 ( gray bar ) with induction of dCas9 SunTag and scFv DNMT3A, yet the methylation level in gene promoter was not affected. CpG DNA methylation level was calculated based on two biological replicates. Error bars represent mean ± s.e.m. of biological replicates. d qPCR shows significant increase of gene expression of DLX1 with induction of dCas9 SunTag and scFv DNMT3A compared to the same cells without induction. P value was computed by two-sided Student’s t-test
    Figure Legend Snippet: Gene-body canyon hypermethylation by dCas9-SunTag-DNMT3A can directly increase oncogene DLX1 expression. a Schematic graph for dCas9-SunTag-DNMT3A DNA methylation editing system. De-activated Cas9 (dCas9) was fused to SunTag epitopes and single-chain variable fragment (scFv) was fused to GFP and DNMT3A to methylate the gene-body canyon and activate gene expression. b Genome browser tracks of gene-body hypermethylated canyons around homeobox gene DLX1 across 30 normal and 35 tumor samples. c CpG DNA methylation level dramatically increased at the gene-body of DLX1 after adding guide RNA DLX1 ( gray bar ) with induction of dCas9 SunTag and scFv DNMT3A, yet the methylation level in gene promoter was not affected. CpG DNA methylation level was calculated based on two biological replicates. Error bars represent mean ± s.e.m. of biological replicates. d qPCR shows significant increase of gene expression of DLX1 with induction of dCas9 SunTag and scFv DNMT3A compared to the same cells without induction. P value was computed by two-sided Student’s t-test

    Techniques Used: Expressing, DNA Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

    59) Product Images from "Evidence against a role for jaagsiekte sheep retrovirus in human lung cancer"

    Article Title: Evidence against a role for jaagsiekte sheep retrovirus in human lung cancer

    Journal: Retrovirology

    doi: 10.1186/s12977-017-0329-6

    PCR detection of JSRV sequences in the genomic DNA of cells exposed to live JSRV virus (LV), heat-inactivated virus (HI) or culture medium alone (−). PCR amplification procedures are described in “ Methods ”. M indicates a 1 kb Plus DNA Ladder (Thermo Fisher) with select band sizes indicated. The expected JSRV amplification product ( arrows ) has a size of 502 bp, which is clearly visible in the live virus (LV) lanes of all cell types except for the NIH 3T3 cells, which lack a functional cell-surface receptor for JSRV. This band is absent in all of the heat inactivated (HI) virus and negative control (−) lanes. The top two panels show an analysis of cellular DNA harvested 3 days after virus exposure, and this experiment was repeated once with identical results. The bottom two panels show an analysis of cellular DNA harvested 8 days after virus exposure, and this experiment was performed once
    Figure Legend Snippet: PCR detection of JSRV sequences in the genomic DNA of cells exposed to live JSRV virus (LV), heat-inactivated virus (HI) or culture medium alone (−). PCR amplification procedures are described in “ Methods ”. M indicates a 1 kb Plus DNA Ladder (Thermo Fisher) with select band sizes indicated. The expected JSRV amplification product ( arrows ) has a size of 502 bp, which is clearly visible in the live virus (LV) lanes of all cell types except for the NIH 3T3 cells, which lack a functional cell-surface receptor for JSRV. This band is absent in all of the heat inactivated (HI) virus and negative control (−) lanes. The top two panels show an analysis of cellular DNA harvested 3 days after virus exposure, and this experiment was repeated once with identical results. The bottom two panels show an analysis of cellular DNA harvested 8 days after virus exposure, and this experiment was performed once

    Techniques Used: Polymerase Chain Reaction, Amplification, Functional Assay, Cell Surface Receptor Assay, Negative Control

    Related Articles

    Amplification:

    Article Title: Single molecule analysis of Trypanosoma brucei DNA replication dynamics
    Article Snippet: Real-time PCR For the real-time PCR analysis after synchronization, genomic DNA was extracted from procyclic T. brucei at the middle and final stages of S phase after release from HU using DNAzol (Invitrogen). .. Segments of 80 bp were amplified from different regions of chromosome 1 using 50 ng of genomic DNA and 50 nM of each primer with a SYBR Green mix (Applied Biosystems) in a final volume of 20 μl.

    Article Title: Development of duplex PCR for differential detection of goatpox and sheeppox viruses
    Article Snippet: The concentration of the purified viral gDNA was measured by NanoDrop 2000 (Thermo Scientific). .. After amplification under the optimized conditions as described above 6 μL PCR amplified products e was tested by nucleic acid electrophoresis, and then was observed by UV gel imaging system.

    SYBR Green Assay:

    Article Title: Single molecule analysis of Trypanosoma brucei DNA replication dynamics
    Article Snippet: Real-time PCR For the real-time PCR analysis after synchronization, genomic DNA was extracted from procyclic T. brucei at the middle and final stages of S phase after release from HU using DNAzol (Invitrogen). .. Segments of 80 bp were amplified from different regions of chromosome 1 using 50 ng of genomic DNA and 50 nM of each primer with a SYBR Green mix (Applied Biosystems) in a final volume of 20 μl.

    Article Title: The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions
    Article Snippet: .. The number of TcRAD51 copies in each strain was determined by using genomic DNA as a template for reactions containing 5 μL SYBR Green PCR Master Mix (Applied Biosystems) with 0.25 M of each primer (TcFwRAD51: GTGCCCTCGTGGTAAACC and TcRevRAD51: GCGGATGAACCCATT). .. Reactions were performed using Applied Biosystems 7900HT Fast Real-Time PCR System.

    Enzymatic Assay:

    Article Title: TALEN mediated somatic mutagenesis in murine models of cancer
    Article Snippet: Genomic DNA for transfected cells and mouse liver samples were collected using QuickExtract DNA Extraction Solution (Thermo Fisher Scientific). .. Genotyping was conducted using PCR, followed by restriction enzyme assay. β-catenin genotyping primers:5′-TTCAGGTAGCATTTTCAGTTCAC-3′ and 5′-GCTAGCTTCCAAACACAAATGC-3′.

    Modification:

    Article Title: The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions
    Article Snippet: Genomic DNA from WT and modified T . cruzi strains were extracted using the PureLink Genomic DNA Mini Kit (ThermoFisher Scientific), using the indicated protocol from the manufacturer. .. The number of TcRAD51 copies in each strain was determined by using genomic DNA as a template for reactions containing 5 μL SYBR Green PCR Master Mix (Applied Biosystems) with 0.25 M of each primer (TcFwRAD51: GTGCCCTCGTGGTAAACC and TcRevRAD51: GCGGATGAACCCATT).

    Transformation Assay:

    Article Title: Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model
    Article Snippet: .. SNP Array Hybridization and DNA Copy Number Analysis DNA from parental SW480 cells, DNA isolated from supernatant of SW480 cells, and genomic DNA from SB1 cells (NIH3T3 passively transformed by SW480 supernatant) was hybridized onto the 500K Affymetrix genotyping array set for DNA copy number analysis, following the manufacturers protocol. .. Analysis was performed using the DNA copy number pipeline in the Partek Genomics Suite software.

    Article Title: Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model
    Article Snippet: .. To gain further insight into this, we hybridized genomic DNA (intracellular) from parental SW480 cells, DNA isolated from supernatant of SW480 cells (extracellular), and genomic DNA from SB1 cells (NIH3T3 passively transformed by SW480 supernatant) onto the 500 K Affymetrix genotyping array set for DNA copy number analysis. ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Single molecule analysis of Trypanosoma brucei DNA replication dynamics
    Article Snippet: Real-time PCR For the real-time PCR analysis after synchronization, genomic DNA was extracted from procyclic T. brucei at the middle and final stages of S phase after release from HU using DNAzol (Invitrogen). .. The following primers were used: ∼397 kb region forward GAA CAA ACG CAT TGG AGG TG and reverse GCA CTT GTT GTC TCC CAA AC; ∼440 kb region forward GTT CCA TGA CTG AGG AGC AG and reverse GTC TCA ACT GGA GGT CGA AG; ∼505 kb region forward CTA CCG ACC GAA AGG AAC TG and reverse CGC TTC AAT CCG AAG CAA AG; ∼550 kb region forward TGG ATG TTC CAC CGC TTT CA and reverse TGT TCT TCA GAT CCT GCG GT; ∼665 kb region forward GGA ATT GGC CCA CAA AAT GG and reverse CAA CAT CAC CGA CTA CCT GG; and ∼798 kb region forward CAA CCG TGA TTC TCT CAG TCA G and reverse CCA CAA AAA TGG TGC CAC AG.

    Derivative Assay:

    Article Title: Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species
    Article Snippet: The custom probe mask files were generated by hybridising gDNA from B. oleracea to Affymetrix A. thaliana ATH-121501 GeneChip® arrays and selecting probe-pairs in which B. oleracea gDNA hybridisation intensity values were greater than 50, 100, 150, 200, 300,...1000. .. The P -values are derived from a one-way ANOVA using a Benjamini and Hochberg False Discovery Rate (0.05) multiple testing correction.

    Hybridization:

    Article Title: Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model
    Article Snippet: .. SNP Array Hybridization and DNA Copy Number Analysis DNA from parental SW480 cells, DNA isolated from supernatant of SW480 cells, and genomic DNA from SB1 cells (NIH3T3 passively transformed by SW480 supernatant) was hybridized onto the 500K Affymetrix genotyping array set for DNA copy number analysis, following the manufacturers protocol. .. Analysis was performed using the DNA copy number pipeline in the Partek Genomics Suite software.

    Article Title: Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species
    Article Snippet: .. The custom probe mask files were generated by hybridising gDNA from B. oleracea to Affymetrix A. thaliana ATH-121501 GeneChip® arrays and selecting probe-pairs in which B. oleracea gDNA hybridisation intensity values were greater than 50, 100, 150, 200, 300,...1000. ..

    Article Title: Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery
    Article Snippet: Southern blot After one month of inoculation with naked plasmid pcDNA3-Hsp65, genomic DNA was isolated from the livers of immunized mice, as well as from those of nonimmunized mice, using Trizol reagent (Invitrogen). .. The Southern blot analysis was carried out using Gene Images™ (Amersham Pharmacia Biotech, Uppsala, Sweden), and hybridization bands were revealed using naked pcDNA3 vector (1 μg/ml) labeled with the random prime labeling module (Amersham).

    Article Title: Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model
    Article Snippet: In any case, the results of our Southern hybridization experiments ( C ) at the very least suggest that transfer may not be limited to a few DNA sequences. .. To gain further insight into this, we hybridized genomic DNA (intracellular) from parental SW480 cells, DNA isolated from supernatant of SW480 cells (extracellular), and genomic DNA from SB1 cells (NIH3T3 passively transformed by SW480 supernatant) onto the 500 K Affymetrix genotyping array set for DNA copy number analysis.

    Transfection:

    Article Title: TALEN mediated somatic mutagenesis in murine models of cancer
    Article Snippet: .. Genomic DNA for transfected cells and mouse liver samples were collected using QuickExtract DNA Extraction Solution (Thermo Fisher Scientific). .. Genotyping was conducted using PCR, followed by restriction enzyme assay. β-catenin genotyping primers:5′-TTCAGGTAGCATTTTCAGTTCAC-3′ and 5′-GCTAGCTTCCAAACACAAATGC-3′.

    Southern Blot:

    Article Title: Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery
    Article Snippet: .. Southern blot After one month of inoculation with naked plasmid pcDNA3-Hsp65, genomic DNA was isolated from the livers of immunized mice, as well as from those of nonimmunized mice, using Trizol reagent (Invitrogen). ..

    Incubation:

    Article Title: C1q/ TNF‐related peptide 8 ( CTRP8) promotes temozolomide resistance in human glioblastoma
    Article Snippet: 2.11 Detection of AP sites in genomic DNA U87MG cells were pretreated with 100 ng·mL−1 of CTRP8 in 1% FBS for 24 h prior to treatment with 1.5 mm TMZ for 15 min. Genomic DNA was extracted using Genomic DNA Mini Kit (Thermo Scientific) according to the manufacturer's instructions. .. Ten microliters of genomic DNA (100 μg·mL−1 ) in TE buffer was incubated with 10 μL of 5 mm ARP solution at 37 °C for 1 h. ARP‐labeled DNA in DNA binding solution was added to a 96‐well plate and incubated at 37 °C overnight in the dark.

    Generated:

    Article Title: Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species
    Article Snippet: .. The custom probe mask files were generated by hybridising gDNA from B. oleracea to Affymetrix A. thaliana ATH-121501 GeneChip® arrays and selecting probe-pairs in which B. oleracea gDNA hybridisation intensity values were greater than 50, 100, 150, 200, 300,...1000. ..

    Imaging:

    Article Title: Development of duplex PCR for differential detection of goatpox and sheeppox viruses
    Article Snippet: The concentration of the purified viral gDNA was measured by NanoDrop 2000 (Thermo Scientific). .. After amplification under the optimized conditions as described above 6 μL PCR amplified products e was tested by nucleic acid electrophoresis, and then was observed by UV gel imaging system.

    Polymerase Chain Reaction:

    Article Title: TALEN mediated somatic mutagenesis in murine models of cancer
    Article Snippet: Genomic DNA for transfected cells and mouse liver samples were collected using QuickExtract DNA Extraction Solution (Thermo Fisher Scientific). .. Genotyping was conducted using PCR, followed by restriction enzyme assay. β-catenin genotyping primers:5′-TTCAGGTAGCATTTTCAGTTCAC-3′ and 5′-GCTAGCTTCCAAACACAAATGC-3′.

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: .. Analytical Performances of the PCR Assays on Genomic MTB DNA Performances of the assays were assessed using genomic DNA from M. tuberculosis mc2 7000 and M. bovis BCG, then were quantified using Qubit® fluorescent dyes quantitation method (Thermo Fisher Scientific); and using clinical specimens. .. Direct smears were prepared from the specimens and stained using the Ziehl-Neelsen and auramine staining method.

    Article Title: The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions
    Article Snippet: .. The number of TcRAD51 copies in each strain was determined by using genomic DNA as a template for reactions containing 5 μL SYBR Green PCR Master Mix (Applied Biosystems) with 0.25 M of each primer (TcFwRAD51: GTGCCCTCGTGGTAAACC and TcRevRAD51: GCGGATGAACCCATT). .. Reactions were performed using Applied Biosystems 7900HT Fast Real-Time PCR System.

    Article Title: Development of duplex PCR for differential detection of goatpox and sheeppox viruses
    Article Snippet: Paragraph title: Duplex PCR sensitivity for detection of GTPV and SPPV ... The concentration of the purified viral gDNA was measured by NanoDrop 2000 (Thermo Scientific).

    Binding Assay:

    Article Title: C1q/ TNF‐related peptide 8 ( CTRP8) promotes temozolomide resistance in human glioblastoma
    Article Snippet: 2.11 Detection of AP sites in genomic DNA U87MG cells were pretreated with 100 ng·mL−1 of CTRP8 in 1% FBS for 24 h prior to treatment with 1.5 mm TMZ for 15 min. Genomic DNA was extracted using Genomic DNA Mini Kit (Thermo Scientific) according to the manufacturer's instructions. .. Ten microliters of genomic DNA (100 μg·mL−1 ) in TE buffer was incubated with 10 μL of 5 mm ARP solution at 37 °C for 1 h. ARP‐labeled DNA in DNA binding solution was added to a 96‐well plate and incubated at 37 °C overnight in the dark.

    Cellular Antioxidant Activity Assay:

    Article Title: Single molecule analysis of Trypanosoma brucei DNA replication dynamics
    Article Snippet: Real-time PCR For the real-time PCR analysis after synchronization, genomic DNA was extracted from procyclic T. brucei at the middle and final stages of S phase after release from HU using DNAzol (Invitrogen). .. The following primers were used: ∼397 kb region forward GAA CAA ACG CAT TGG AGG TG and reverse GCA CTT GTT GTC TCC CAA AC; ∼440 kb region forward GTT CCA TGA CTG AGG AGC AG and reverse GTC TCA ACT GGA GGT CGA AG; ∼505 kb region forward CTA CCG ACC GAA AGG AAC TG and reverse CGC TTC AAT CCG AAG CAA AG; ∼550 kb region forward TGG ATG TTC CAC CGC TTT CA and reverse TGT TCT TCA GAT CCT GCG GT; ∼665 kb region forward GGA ATT GGC CCA CAA AAT GG and reverse CAA CAT CAC CGA CTA CCT GG; and ∼798 kb region forward CAA CCG TGA TTC TCT CAG TCA G and reverse CCA CAA AAA TGG TGC CAC AG.

    DNA Extraction:

    Article Title: TALEN mediated somatic mutagenesis in murine models of cancer
    Article Snippet: .. Genomic DNA for transfected cells and mouse liver samples were collected using QuickExtract DNA Extraction Solution (Thermo Fisher Scientific). .. Genotyping was conducted using PCR, followed by restriction enzyme assay. β-catenin genotyping primers:5′-TTCAGGTAGCATTTTCAGTTCAC-3′ and 5′-GCTAGCTTCCAAACACAAATGC-3′.

    Nucleic Acid Electrophoresis:

    Article Title: Development of duplex PCR for differential detection of goatpox and sheeppox viruses
    Article Snippet: The concentration of the purified viral gDNA was measured by NanoDrop 2000 (Thermo Scientific). .. After amplification under the optimized conditions as described above 6 μL PCR amplified products e was tested by nucleic acid electrophoresis, and then was observed by UV gel imaging system.

    Isolation:

    Article Title: Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model
    Article Snippet: .. SNP Array Hybridization and DNA Copy Number Analysis DNA from parental SW480 cells, DNA isolated from supernatant of SW480 cells, and genomic DNA from SB1 cells (NIH3T3 passively transformed by SW480 supernatant) was hybridized onto the 500K Affymetrix genotyping array set for DNA copy number analysis, following the manufacturers protocol. .. Analysis was performed using the DNA copy number pipeline in the Partek Genomics Suite software.

    Article Title: Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery
    Article Snippet: .. Southern blot After one month of inoculation with naked plasmid pcDNA3-Hsp65, genomic DNA was isolated from the livers of immunized mice, as well as from those of nonimmunized mice, using Trizol reagent (Invitrogen). ..

    Article Title: Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model
    Article Snippet: .. To gain further insight into this, we hybridized genomic DNA (intracellular) from parental SW480 cells, DNA isolated from supernatant of SW480 cells (extracellular), and genomic DNA from SB1 cells (NIH3T3 passively transformed by SW480 supernatant) onto the 500 K Affymetrix genotyping array set for DNA copy number analysis. ..

    Labeling:

    Article Title: Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery
    Article Snippet: Southern blot After one month of inoculation with naked plasmid pcDNA3-Hsp65, genomic DNA was isolated from the livers of immunized mice, as well as from those of nonimmunized mice, using Trizol reagent (Invitrogen). .. The Southern blot analysis was carried out using Gene Images™ (Amersham Pharmacia Biotech, Uppsala, Sweden), and hybridization bands were revealed using naked pcDNA3 vector (1 μg/ml) labeled with the random prime labeling module (Amersham).

    Article Title: C1q/ TNF‐related peptide 8 ( CTRP8) promotes temozolomide resistance in human glioblastoma
    Article Snippet: 2.11 Detection of AP sites in genomic DNA U87MG cells were pretreated with 100 ng·mL−1 of CTRP8 in 1% FBS for 24 h prior to treatment with 1.5 mm TMZ for 15 min. Genomic DNA was extracted using Genomic DNA Mini Kit (Thermo Scientific) according to the manufacturer's instructions. .. The ARP labeling and quantification of AP sites were performed by AP sites assay kit (Dojindo Molecular Technologies, Burlington, ON, Canada).

    Mouse Assay:

    Article Title: Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery
    Article Snippet: .. Southern blot After one month of inoculation with naked plasmid pcDNA3-Hsp65, genomic DNA was isolated from the livers of immunized mice, as well as from those of nonimmunized mice, using Trizol reagent (Invitrogen). ..

    Sequencing:

    Article Title: Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model
    Article Snippet: Gene Copy Number is Nearly Identical between Intracellular and Extracellular DNA Compartments Sequencing analyses, indicate that circulating DNA comes from the whole genome, with minimal indications of sequence clustering, although there is incomplete coverage of all chromosomes . .. To gain further insight into this, we hybridized genomic DNA (intracellular) from parental SW480 cells, DNA isolated from supernatant of SW480 cells (extracellular), and genomic DNA from SB1 cells (NIH3T3 passively transformed by SW480 supernatant) onto the 500 K Affymetrix genotyping array set for DNA copy number analysis.

    Activated Clotting Time Assay:

    Article Title: Single molecule analysis of Trypanosoma brucei DNA replication dynamics
    Article Snippet: Real-time PCR For the real-time PCR analysis after synchronization, genomic DNA was extracted from procyclic T. brucei at the middle and final stages of S phase after release from HU using DNAzol (Invitrogen). .. The following primers were used: ∼397 kb region forward GAA CAA ACG CAT TGG AGG TG and reverse GCA CTT GTT GTC TCC CAA AC; ∼440 kb region forward GTT CCA TGA CTG AGG AGC AG and reverse GTC TCA ACT GGA GGT CGA AG; ∼505 kb region forward CTA CCG ACC GAA AGG AAC TG and reverse CGC TTC AAT CCG AAG CAA AG; ∼550 kb region forward TGG ATG TTC CAC CGC TTT CA and reverse TGT TCT TCA GAT CCT GCG GT; ∼665 kb region forward GGA ATT GGC CCA CAA AAT GG and reverse CAA CAT CAC CGA CTA CCT GG; and ∼798 kb region forward CAA CCG TGA TTC TCT CAG TCA G and reverse CCA CAA AAA TGG TGC CAC AG.

    Purification:

    Article Title: Development of duplex PCR for differential detection of goatpox and sheeppox viruses
    Article Snippet: .. The concentration of the purified viral gDNA was measured by NanoDrop 2000 (Thermo Scientific). ..

    Plasmid Preparation:

    Article Title: Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery
    Article Snippet: .. Southern blot After one month of inoculation with naked plasmid pcDNA3-Hsp65, genomic DNA was isolated from the livers of immunized mice, as well as from those of nonimmunized mice, using Trizol reagent (Invitrogen). ..

    Software:

    Article Title: Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model
    Article Snippet: SNP Array Hybridization and DNA Copy Number Analysis DNA from parental SW480 cells, DNA isolated from supernatant of SW480 cells, and genomic DNA from SB1 cells (NIH3T3 passively transformed by SW480 supernatant) was hybridized onto the 500K Affymetrix genotyping array set for DNA copy number analysis, following the manufacturers protocol. .. Analysis was performed using the DNA copy number pipeline in the Partek Genomics Suite software.

    Article Title: Successful use of whole genome amplified DNA from multiple source types for high-density Illumina SNP microarrays
    Article Snippet: Identifiler 2 ng of each gDNA and wgaDNA sample (as determined by PicoGreen®) was used as template for the AmpFl STR® Identifiler® assay (Life Technologies) according to manufacturer’s protocol. .. Alleles were scored using GeneMapper 4.0 software with a peak height threshold of 200 RFUs.

    Real-time Polymerase Chain Reaction:

    Article Title: Single molecule analysis of Trypanosoma brucei DNA replication dynamics
    Article Snippet: .. Real-time PCR For the real-time PCR analysis after synchronization, genomic DNA was extracted from procyclic T. brucei at the middle and final stages of S phase after release from HU using DNAzol (Invitrogen). .. Segments of 80 bp were amplified from different regions of chromosome 1 using 50 ng of genomic DNA and 50 nM of each primer with a SYBR Green mix (Applied Biosystems) in a final volume of 20 μl.

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: Analytical Performances of the PCR Assays on Genomic MTB DNA Performances of the assays were assessed using genomic DNA from M. tuberculosis mc2 7000 and M. bovis BCG, then were quantified using Qubit® fluorescent dyes quantitation method (Thermo Fisher Scientific); and using clinical specimens. .. The linear dynamic range of the qPCR assays, variability inter and intra run were evaluated by plotting separately the results of 10 replicates of a 10-fold serial dilutions using the M. tuberculosis H37Rv commercial standard, Mycobacterium bovis BCG DNA and M. tuberculosis mc2 7000 DNA.

    Article Title: The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions
    Article Snippet: Paragraph title: Real-time PCR ... The number of TcRAD51 copies in each strain was determined by using genomic DNA as a template for reactions containing 5 μL SYBR Green PCR Master Mix (Applied Biosystems) with 0.25 M of each primer (TcFwRAD51: GTGCCCTCGTGGTAAACC and TcRevRAD51: GCGGATGAACCCATT).

    Selection:

    Article Title: Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species
    Article Snippet: Raw data were analysed with the RMA pre-normalisation algorithm using the ATH1-121501 probe mask file (no probe selection) or a custom probe mask file. .. The custom probe mask files were generated by hybridising gDNA from B. oleracea to Affymetrix A. thaliana ATH-121501 GeneChip® arrays and selecting probe-pairs in which B. oleracea gDNA hybridisation intensity values were greater than 50, 100, 150, 200, 300,...1000.

    Agarose Gel Electrophoresis:

    Article Title: Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery
    Article Snippet: Southern blot After one month of inoculation with naked plasmid pcDNA3-Hsp65, genomic DNA was isolated from the livers of immunized mice, as well as from those of nonimmunized mice, using Trizol reagent (Invitrogen). .. From each, 10 μg of total cellular DNA were subjected to electrophoresis on a 0.8% agarose gel.

    Electrophoresis:

    Article Title: Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery
    Article Snippet: Southern blot After one month of inoculation with naked plasmid pcDNA3-Hsp65, genomic DNA was isolated from the livers of immunized mice, as well as from those of nonimmunized mice, using Trizol reagent (Invitrogen). .. From each, 10 μg of total cellular DNA were subjected to electrophoresis on a 0.8% agarose gel.

    Article Title: The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions
    Article Snippet: DNA integrity was verified by running the samples onto an electrophoresis gel, and 5 ng of total DNA were used for real-time PCR. .. The number of TcRAD51 copies in each strain was determined by using genomic DNA as a template for reactions containing 5 μL SYBR Green PCR Master Mix (Applied Biosystems) with 0.25 M of each primer (TcFwRAD51: GTGCCCTCGTGGTAAACC and TcRevRAD51: GCGGATGAACCCATT).

    Quantitation Assay:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: .. Analytical Performances of the PCR Assays on Genomic MTB DNA Performances of the assays were assessed using genomic DNA from M. tuberculosis mc2 7000 and M. bovis BCG, then were quantified using Qubit® fluorescent dyes quantitation method (Thermo Fisher Scientific); and using clinical specimens. .. Direct smears were prepared from the specimens and stained using the Ziehl-Neelsen and auramine staining method.

    Spectrophotometry:

    Article Title: Novel extraction of high quality genomic DNA from frozen bovine blood samples by using detergent method
    Article Snippet: .. Quality and quantity measurement of extracted gDNA The quality and quantity of the gDNA extracted by PCI and Detergent methods were assessed by using a Nano-Drop Spectrophotometer (Shimadzu BioSpec-nano® Japan). ..

    Concentration Assay:

    Article Title: Successful use of whole genome amplified DNA from multiple source types for high-density Illumina SNP microarrays
    Article Snippet: Quantification Both gDNA and wgaDNA samples were quantified using the Quant-iT™ PicoGreen® dsDNA Reagent (Life Technologies) according to manufacturer’s protocol. .. All samples were normalized to desired concentration for input into Illumina® Infinium® LCG assay which is 50 ng/μL.

    Article Title: Novel extraction of high quality genomic DNA from frozen bovine blood samples by using detergent method
    Article Snippet: Quality and quantity measurement of extracted gDNA The quality and quantity of the gDNA extracted by PCI and Detergent methods were assessed by using a Nano-Drop Spectrophotometer (Shimadzu BioSpec-nano® Japan). .. Good concentration of gDNA was associated with better gDNA quantity and purity.

    Article Title: Development of duplex PCR for differential detection of goatpox and sheeppox viruses
    Article Snippet: .. The concentration of the purified viral gDNA was measured by NanoDrop 2000 (Thermo Scientific). ..

    CTG Assay:

    Article Title: Single molecule analysis of Trypanosoma brucei DNA replication dynamics
    Article Snippet: Real-time PCR For the real-time PCR analysis after synchronization, genomic DNA was extracted from procyclic T. brucei at the middle and final stages of S phase after release from HU using DNAzol (Invitrogen). .. The following primers were used: ∼397 kb region forward GAA CAA ACG CAT TGG AGG TG and reverse GCA CTT GTT GTC TCC CAA AC; ∼440 kb region forward GTT CCA TGA CTG AGG AGC AG and reverse GTC TCA ACT GGA GGT CGA AG; ∼505 kb region forward CTA CCG ACC GAA AGG AAC TG and reverse CGC TTC AAT CCG AAG CAA AG; ∼550 kb region forward TGG ATG TTC CAC CGC TTT CA and reverse TGT TCT TCA GAT CCT GCG GT; ∼665 kb region forward GGA ATT GGC CCA CAA AAT GG and reverse CAA CAT CAC CGA CTA CCT GG; and ∼798 kb region forward CAA CCG TGA TTC TCT CAG TCA G and reverse CCA CAA AAA TGG TGC CAC AG.

    Staining:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: Analytical Performances of the PCR Assays on Genomic MTB DNA Performances of the assays were assessed using genomic DNA from M. tuberculosis mc2 7000 and M. bovis BCG, then were quantified using Qubit® fluorescent dyes quantitation method (Thermo Fisher Scientific); and using clinical specimens. .. Direct smears were prepared from the specimens and stained using the Ziehl-Neelsen and auramine staining method.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Thermo Fisher magmax total nucleic acid isolation kit
    Magmax Total Nucleic Acid Isolation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/magmax total nucleic acid isolation kit/product/Thermo Fisher
    Average 90 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    magmax total nucleic acid isolation kit - by Bioz Stars, 2020-01
    90/100 stars
      Buy from Supplier

    N/A
    The iPrep Protocol Card is a flash 512 KB memory card Each iPrep Protocol card is pre programmed with the purification protocol that directs the volume of reagents used and
      Buy from Supplier

    N/A
    Thermo Scientific GeneJET Plant gDNA Binding Solution is a component of the GeneJET Plant RNA Purification Mini Kit K0791 K0792 and may be purchased separately Provided in 40 mL aliquots
      Buy from Supplier

    Image Search Results