Structured Review

Thermo Fisher pcr mix
RT-qPCR standard curves. Standard curve relating viral copy number to Ct value from RT-Polymerase chain reaction using 10-fold dilutions of in-vitro transcribed RNA using (a) SYBR ® Green I and (b) <t>TaqMan</t> chemistries.
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1) Product Images from "Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology"

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology

Journal: PLoS ONE

doi: 10.1371/journal.pone.0159155

RT-qPCR standard curves. Standard curve relating viral copy number to Ct value from RT-Polymerase chain reaction using 10-fold dilutions of in-vitro transcribed RNA using (a) SYBR ® Green I and (b) TaqMan chemistries.
Figure Legend Snippet: RT-qPCR standard curves. Standard curve relating viral copy number to Ct value from RT-Polymerase chain reaction using 10-fold dilutions of in-vitro transcribed RNA using (a) SYBR ® Green I and (b) TaqMan chemistries.

Techniques Used: Quantitative RT-PCR, Polymerase Chain Reaction, In Vitro, SYBR Green Assay

2) Product Images from "The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis"

Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erw181

Functional characterization of VvibZIPC22 in tobacco overexpressing lines. (A) Stamens and corollas of wild-type (WT) plants and transgenic lines (L), and (B) average VvibZIPC22 relative expression in stamens (S) and petal limbs (P) of three WT and six L at two developmental stages (S1=open flower without pollen, S2= mature flower). (C) Variation of cyanidin 3-rutinoside (Cyan 3-rut), quercetin 3-rutinoside (Que 3-rut), and kaempferol 3-rutinoside (Kaempf 3-rut) in stamens and of proanthocyanidins (PAs) in petal limbs of L and WT plants at the two stages. Anthocyanin and flavonol contents were determined by HPLC-DAD, while PA content was determined by DMACA. (D) Fold induction in gene expression of tobacco flavonoid structural genes between L and WT stamens and petal limbs. Transcript levels in both (B) and (D) were determined by qRT-PCR using gene-specific primers ( Supplementary Table S1 ), calibration against the average expression value in all the lines at each stage, and normalization against NteIF4a and NtUbiquitin relative expression. Average values with error bars indicating the SE are shown. Asterisks indicate significant changes ( P
Figure Legend Snippet: Functional characterization of VvibZIPC22 in tobacco overexpressing lines. (A) Stamens and corollas of wild-type (WT) plants and transgenic lines (L), and (B) average VvibZIPC22 relative expression in stamens (S) and petal limbs (P) of three WT and six L at two developmental stages (S1=open flower without pollen, S2= mature flower). (C) Variation of cyanidin 3-rutinoside (Cyan 3-rut), quercetin 3-rutinoside (Que 3-rut), and kaempferol 3-rutinoside (Kaempf 3-rut) in stamens and of proanthocyanidins (PAs) in petal limbs of L and WT plants at the two stages. Anthocyanin and flavonol contents were determined by HPLC-DAD, while PA content was determined by DMACA. (D) Fold induction in gene expression of tobacco flavonoid structural genes between L and WT stamens and petal limbs. Transcript levels in both (B) and (D) were determined by qRT-PCR using gene-specific primers ( Supplementary Table S1 ), calibration against the average expression value in all the lines at each stage, and normalization against NteIF4a and NtUbiquitin relative expression. Average values with error bars indicating the SE are shown. Asterisks indicate significant changes ( P

Techniques Used: Functional Assay, Transgenic Assay, Expressing, High Performance Liquid Chromatography, Quantitative RT-PCR

(A) Profiles of VvibZIPC22 relative expression and of six flavonol aglycons at different Pinot Noir berry developmental stages. (B) VvibZIPC22 relative expression in a panel of 15 Pinot Noir organs. Transcript levels (NRQs) were determined by qRT-PCR using gene-specific primers ( Supplementary Table S1 ), calibration against the mean expression value in all the stages, and normalization against VviGADPH and VviACTIN relative expression. Flavonol content was determined by HPLC-DAD analysis. Each value corresponds to the mean and SE of three different biological replicates. F, flowering (50% opened flowers, E-L 23); P, pepper corn (E-L 29); PV, pre-véraison (hard green berries, E-L 33); V, véraison (50% coloured berries, E-L 35); 16°, post-véraison (berries at 16° Brix, E-L 36); 18°, maturity (berries at 18° Brix, E-L 38); NRQ, normalized relative quantity; FW, fresh weight.
Figure Legend Snippet: (A) Profiles of VvibZIPC22 relative expression and of six flavonol aglycons at different Pinot Noir berry developmental stages. (B) VvibZIPC22 relative expression in a panel of 15 Pinot Noir organs. Transcript levels (NRQs) were determined by qRT-PCR using gene-specific primers ( Supplementary Table S1 ), calibration against the mean expression value in all the stages, and normalization against VviGADPH and VviACTIN relative expression. Flavonol content was determined by HPLC-DAD analysis. Each value corresponds to the mean and SE of three different biological replicates. F, flowering (50% opened flowers, E-L 23); P, pepper corn (E-L 29); PV, pre-véraison (hard green berries, E-L 33); V, véraison (50% coloured berries, E-L 35); 16°, post-véraison (berries at 16° Brix, E-L 36); 18°, maturity (berries at 18° Brix, E-L 38); NRQ, normalized relative quantity; FW, fresh weight.

Techniques Used: Expressing, Quantitative RT-PCR, High Performance Liquid Chromatography

UV light responsiveness of VvibZIPC22 and flavonol pathway genes. (A) Induction of VvibZIPC22 , VviFLS1 , and VviMYBF1 in Chardonnay leaves after UV light treatment. Each bar corresponds to the fold induction in gene expression between treated and control leaves as determined by qRT-PCR. Transcript levels were measured using gene-specific primers ( Supplementary Table S1 ), calibration against the expression value in the control sample at 0h post-treatment, and normalization against VviGADPH and VviUbiquitin relative expression. (B) Accumulation of flavonols in Chardonnay leaves after UV light treatment. Each bar corresponds to the fold induction in the total content of flavonol aglycons between treated and control leaves as detected by HPLC-DAD analysis. For both measurements, each data point corresponds to the mean and SE of three independent extractions of the same biological material. (C) Putative light regulatory elements identified in the promoter of VvibZIPC22 (PN, Ch) in this study (underlined) and in the promoters of VviMYBF1 and VviFLS1 ( Czemmel et al. , 2009 ). These cis -elements were aligned with the corresponding Arabidopsis elements taken from the PLACE database ( Higo et al. , 1999 ) using GeneDoc software (v. 1.6). Nucleotides are labelled from black to light grey according to their conservation. Numbers in front of the alignment indicate the relative distance of each element from the putative transcriptional start site (+1). CHS, chalcone synthase; FLS, flavonol synthase; PN, Pinot Noir; Ch, Chardonnay; ACS, ACGT-containing sequence similar to ACE in the Arabidopsis CHS promoter (accession no. S000355); MRS, MYB recognition sequence similar to MRE in the Arabidopsis CHS promoter (S000356); MYBCORE (S000176), IBOXCORE element (S000199); RRS, R response sequence similar to RRE in the Arabidopsis CHS promoter (S000407).
Figure Legend Snippet: UV light responsiveness of VvibZIPC22 and flavonol pathway genes. (A) Induction of VvibZIPC22 , VviFLS1 , and VviMYBF1 in Chardonnay leaves after UV light treatment. Each bar corresponds to the fold induction in gene expression between treated and control leaves as determined by qRT-PCR. Transcript levels were measured using gene-specific primers ( Supplementary Table S1 ), calibration against the expression value in the control sample at 0h post-treatment, and normalization against VviGADPH and VviUbiquitin relative expression. (B) Accumulation of flavonols in Chardonnay leaves after UV light treatment. Each bar corresponds to the fold induction in the total content of flavonol aglycons between treated and control leaves as detected by HPLC-DAD analysis. For both measurements, each data point corresponds to the mean and SE of three independent extractions of the same biological material. (C) Putative light regulatory elements identified in the promoter of VvibZIPC22 (PN, Ch) in this study (underlined) and in the promoters of VviMYBF1 and VviFLS1 ( Czemmel et al. , 2009 ). These cis -elements were aligned with the corresponding Arabidopsis elements taken from the PLACE database ( Higo et al. , 1999 ) using GeneDoc software (v. 1.6). Nucleotides are labelled from black to light grey according to their conservation. Numbers in front of the alignment indicate the relative distance of each element from the putative transcriptional start site (+1). CHS, chalcone synthase; FLS, flavonol synthase; PN, Pinot Noir; Ch, Chardonnay; ACS, ACGT-containing sequence similar to ACE in the Arabidopsis CHS promoter (accession no. S000355); MRS, MYB recognition sequence similar to MRE in the Arabidopsis CHS promoter (S000356); MYBCORE (S000176), IBOXCORE element (S000199); RRS, R response sequence similar to RRE in the Arabidopsis CHS promoter (S000407).

Techniques Used: Expressing, Quantitative RT-PCR, High Performance Liquid Chromatography, Software, Sequencing

3) Product Images from "Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT"

Article Title: Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT

Journal: British Journal of Cancer

doi: 10.1038/bjc.2013.168

MRP-1 splicing events observed in ESFT primary tissues. Splicing events observed in primary ESFTs (sample 40) generated by RT-PCR, employing 11 primer mixes spanning the 31 exons of MRP-1 ( Supplementary Figure 1 ). Image shows ethidium bromide-stained amplified products after separation by electrophoresis and visualisation under UV light. *=splice variant; ←=full-length/predicted PCR product; ladder=50 bp DNA ladder;+=RT-positive amplified sample; – =RT-negative control; +H 2 O=control for contamination containing RT, but in which RNA is replaced with H 2 O; H 2 O=negative control in which cDNA is replaced with H 2 O in PCR step.
Figure Legend Snippet: MRP-1 splicing events observed in ESFT primary tissues. Splicing events observed in primary ESFTs (sample 40) generated by RT-PCR, employing 11 primer mixes spanning the 31 exons of MRP-1 ( Supplementary Figure 1 ). Image shows ethidium bromide-stained amplified products after separation by electrophoresis and visualisation under UV light. *=splice variant; ←=full-length/predicted PCR product; ladder=50 bp DNA ladder;+=RT-positive amplified sample; – =RT-negative control; +H 2 O=control for contamination containing RT, but in which RNA is replaced with H 2 O; H 2 O=negative control in which cDNA is replaced with H 2 O in PCR step.

Techniques Used: Generated, Reverse Transcription Polymerase Chain Reaction, Staining, Amplification, Electrophoresis, Variant Assay, Polymerase Chain Reaction, Negative Control

4) Product Images from "Peruvian Maca (Lepidium peruvianum): (I) Phytochemical and Genetic Differences in Three Maca Phenotypes"

Article Title: Peruvian Maca (Lepidium peruvianum): (I) Phytochemical and Genetic Differences in Three Maca Phenotypes

Journal: International Journal of Biomedical Science : IJBS

doi:

DNA extracted from three fresh Maca hypocotyls ( L. peruvianum Chacon) Lb = Black; Lcz = Red; Lz = Yellow. Lines observed in RAPD analysis, particularly in wells 4 (Figure 5a: primer A-02 5'-TGCCGAGCTG-3') and well 57 (4th from the right - Figure 5b: primer A-19 5'-CAAACGTCGG-3') pointing with arrows at phenotypes Black (Lb) and Yellow (Lz) respectively may indicate an existence of genetic polymorphism. *Boxes above each of the three phenotypes sequence (Lb, Lcz and Lz) indicate well numbers (from – to) and corresponding primer code used for the sequence. The reaction used: 50 ng DNA, 20 pmol of each primer (OPL Kit OPERON or single RAPD primer OLIGO), and 7.5 μl of PCR Mix (Fermentas 2×). The final volume of the reactions was 15 μl.
Figure Legend Snippet: DNA extracted from three fresh Maca hypocotyls ( L. peruvianum Chacon) Lb = Black; Lcz = Red; Lz = Yellow. Lines observed in RAPD analysis, particularly in wells 4 (Figure 5a: primer A-02 5'-TGCCGAGCTG-3') and well 57 (4th from the right - Figure 5b: primer A-19 5'-CAAACGTCGG-3') pointing with arrows at phenotypes Black (Lb) and Yellow (Lz) respectively may indicate an existence of genetic polymorphism. *Boxes above each of the three phenotypes sequence (Lb, Lcz and Lz) indicate well numbers (from – to) and corresponding primer code used for the sequence. The reaction used: 50 ng DNA, 20 pmol of each primer (OPL Kit OPERON or single RAPD primer OLIGO), and 7.5 μl of PCR Mix (Fermentas 2×). The final volume of the reactions was 15 μl.

Techniques Used: Sequencing, Polymerase Chain Reaction

5) Product Images from "OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics"

Article Title: OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics

Journal: Journal of Immunology Research

doi: 10.1155/2018/2505818

Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.
Figure Legend Snippet: Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Western Blot, Amplification, Agarose Gel Electrophoresis, Staining, Knock-Out, Expressing

6) Product Images from "Phylogenetic Analysis of Bacillus cereus sensu lato Isolates from Commercial Bee Pollen Using tRNACys-PCR"

Article Title: Phylogenetic Analysis of Bacillus cereus sensu lato Isolates from Commercial Bee Pollen Using tRNACys-PCR

Journal: Microorganisms

doi: 10.3390/microorganisms8040524

B. cereus s.l. detection in commercial bee pollen. ( A ) Panel A show positive results of the tRNA Cys -PCR analyses for isolates D8 to D14 and ( B ) panel B show negative results for tRNA Cys -PCR (see text for bacterial species identified for lanes 1 to 6). Lane M contains a Thermo Scientific GeneRuler 1 Kb DNA ladder, lane C is the control ( B. cereus ATCC 10876), and the arrows show the PCR amplification product. ( C ) Bacterial species identified by MALDI-TOF MS; numbers in parentheses indicate the total number of isolated colonies identified.
Figure Legend Snippet: B. cereus s.l. detection in commercial bee pollen. ( A ) Panel A show positive results of the tRNA Cys -PCR analyses for isolates D8 to D14 and ( B ) panel B show negative results for tRNA Cys -PCR (see text for bacterial species identified for lanes 1 to 6). Lane M contains a Thermo Scientific GeneRuler 1 Kb DNA ladder, lane C is the control ( B. cereus ATCC 10876), and the arrows show the PCR amplification product. ( C ) Bacterial species identified by MALDI-TOF MS; numbers in parentheses indicate the total number of isolated colonies identified.

Techniques Used: Polymerase Chain Reaction, Amplification, Isolation

7) Product Images from "Gene expression analysis of ischaemia and reperfusion in human microsurgical free muscle tissue transfer"

Article Title: Gene expression analysis of ischaemia and reperfusion in human microsurgical free muscle tissue transfer

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2010.01061.x

Real-time-quantitative-PCR reveals increased expression of Caspase-8. (A) Interleukin-8 (B), PLAUR (C) and S100A8 (D).
Figure Legend Snippet: Real-time-quantitative-PCR reveals increased expression of Caspase-8. (A) Interleukin-8 (B), PLAUR (C) and S100A8 (D).

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

Exemplary DNA gel electrophoresis of real-time-quantitative-PCR product of patient 1.
Figure Legend Snippet: Exemplary DNA gel electrophoresis of real-time-quantitative-PCR product of patient 1.

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

8) Product Images from "MLGA--a rapid and cost-efficient assay for gene copy-number analysis"

Article Title: MLGA--a rapid and cost-efficient assay for gene copy-number analysis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm651

( a ) Multiplex ligation-dependent genome amplification (MLGA), reaction scheme. (I) Genomic DNA is digested by restriction enzyme to generate targets with defined ends. (II) Each MLGA probe consists of two oligonucleotides, one selector oligo of 70–74 nt (green) and one general vector oligo of 34 nt (red). MLGA probe together with DNA-ligase forms circular DNA of target molecules after denaturation and hybridization. (III) To reduce background signal in the assay, undesirable, linear DNA is degraded by exonuclease I (Exo I). (IV) Multiplex PCR is facilitated by using universal primers that hybridize to a sequence in the vector. PCR products are analyzed using the Agilent Bioanalyzer 2100™ electrophoresis system. ( b ) Data from an MLGA set of 14 probes targeting loci on human chromosomes 13, 18, 21, X and Y. The upper graph shows the resulting elution diagrams from analyses of male and female DNA pools.
Figure Legend Snippet: ( a ) Multiplex ligation-dependent genome amplification (MLGA), reaction scheme. (I) Genomic DNA is digested by restriction enzyme to generate targets with defined ends. (II) Each MLGA probe consists of two oligonucleotides, one selector oligo of 70–74 nt (green) and one general vector oligo of 34 nt (red). MLGA probe together with DNA-ligase forms circular DNA of target molecules after denaturation and hybridization. (III) To reduce background signal in the assay, undesirable, linear DNA is degraded by exonuclease I (Exo I). (IV) Multiplex PCR is facilitated by using universal primers that hybridize to a sequence in the vector. PCR products are analyzed using the Agilent Bioanalyzer 2100™ electrophoresis system. ( b ) Data from an MLGA set of 14 probes targeting loci on human chromosomes 13, 18, 21, X and Y. The upper graph shows the resulting elution diagrams from analyses of male and female DNA pools.

Techniques Used: Multiplex Assay, Ligation, Amplification, Plasmid Preparation, Hybridization, Polymerase Chain Reaction, Sequencing, Electrophoresis

9) Product Images from "Bovine Papillomavirus Type 13 DNA in Equine Sarcoids"

Article Title: Bovine Papillomavirus Type 13 DNA in Equine Sarcoids

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.00371-13

PCR amplification of partial fragments of the L1 and E1 genes.
Figure Legend Snippet: PCR amplification of partial fragments of the L1 and E1 genes.

Techniques Used: Polymerase Chain Reaction, Amplification

10) Product Images from "Expression of Arabidopsis MYB transcription factor, AtMYB111, in tobacco requires light to modulate flavonol content"

Article Title: Expression of Arabidopsis MYB transcription factor, AtMYB111, in tobacco requires light to modulate flavonol content

Journal: Scientific Reports

doi: 10.1038/srep05018

Development of AtMYB111 -expressing transgenic tobacco lines and their phenotypic characterization. (a) Schematic representation of T-DNA region of plant expression construct carrying AtMYB111 cDNA in pBI121 vector used for tobacco transformation. (b) Flower color alterations in different transgenic lines. The pigmentation in petals of transgenic lines expressing AtMYB111 (1, 2 and 3) was compared with WT and empty vector (EV) transformed tobacco lines. (c) Confirmation of the presence of the transgene by PCR amplification of transgene using forward and reverse primers of AtMYB111 in different transgenic lines. (d) Expression analysis of transgene through semiquantitative RT-PCR using total RNA from leaves of WT, EV and different transgenic lines. (e) Total anthocyanin content in petals of WT, EV and different transgenic lines. Data are expressed as means ± SE of at least 3 independent experiments, each experiment consisting of 3 technical replicates. In (c) and (d), gels have been cropped for clarity and conciseness of the presentation. *** indicate values that differ significantly from WT at P
Figure Legend Snippet: Development of AtMYB111 -expressing transgenic tobacco lines and their phenotypic characterization. (a) Schematic representation of T-DNA region of plant expression construct carrying AtMYB111 cDNA in pBI121 vector used for tobacco transformation. (b) Flower color alterations in different transgenic lines. The pigmentation in petals of transgenic lines expressing AtMYB111 (1, 2 and 3) was compared with WT and empty vector (EV) transformed tobacco lines. (c) Confirmation of the presence of the transgene by PCR amplification of transgene using forward and reverse primers of AtMYB111 in different transgenic lines. (d) Expression analysis of transgene through semiquantitative RT-PCR using total RNA from leaves of WT, EV and different transgenic lines. (e) Total anthocyanin content in petals of WT, EV and different transgenic lines. Data are expressed as means ± SE of at least 3 independent experiments, each experiment consisting of 3 technical replicates. In (c) and (d), gels have been cropped for clarity and conciseness of the presentation. *** indicate values that differ significantly from WT at P

Techniques Used: Expressing, Transgenic Assay, Construct, Plasmid Preparation, Transformation Assay, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction

11) Product Images from "Induction of IFN-α Subtypes and Their Antiviral Activity in Mumps Virus Infection"

Article Title: Induction of IFN-α Subtypes and Their Antiviral Activity in Mumps Virus Infection

Journal: Viral Immunology

doi: 10.1089/vim.2014.0028

Induction of IFNB1 and IFNL1 genes in cells infected with MuV. Relative expression of mRNA was measured at 8, 24, 48, and 72 h after infection of MRC-5 (A) or A549 cells (B) by quantitative PCR. Obtained values were normalized against housekeeping
Figure Legend Snippet: Induction of IFNB1 and IFNL1 genes in cells infected with MuV. Relative expression of mRNA was measured at 8, 24, 48, and 72 h after infection of MRC-5 (A) or A549 cells (B) by quantitative PCR. Obtained values were normalized against housekeeping

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

12) Product Images from "Correlation between Point Mutations in NS2 and the Viability and Cytopathogenicity of Bovine Viral Diarrhea Virus Strain Oregon Analyzed with an Infectious cDNA Clone"

Article Title: Correlation between Point Mutations in NS2 and the Viability and Cytopathogenicity of Bovine Viral Diarrhea Virus Strain Oregon Analyzed with an Infectious cDNA Clone

Journal: Journal of Virology

doi:

Analyses following the transfection of RNA derived from pOR with the S codon at position 1555 of the ORF exchanged for an F codon. (A) Detection of cells containing replicating virus resulting from RNA transfection. Cells were transfected with equal doses of the in vitro-transcribed RNAs by the DEAE-dextran method and seeded at a sufficient density that 3 days posttransfection a closed layer of cells was obtained. At this time point, cells were fixed and virus-containing cells were visualized by MAb-mediated peroxidase staining. Below the dishes, the NS2-3 cleavage efficiency determined for the respective polyprotein after transient expression is indicated. (B) Analysis of part of the NS2 sequence from RNA obtained at different time points after transfection of RNA derived from pOR/S-F. Cells were split every 3 to 4 days after transfection, and total RNA was prepared from each passage and used as starting material for RT-PCR. RNA from passage 0 was isolated 3 days posttransfection. The amplified fragments were directly sequenced to look for the mutation introduced into pOR/S-F (arrowhead). In lanes C, the sequence of the RT-PCR product derived from the in vitro-transcribed RNA is shown as a control.
Figure Legend Snippet: Analyses following the transfection of RNA derived from pOR with the S codon at position 1555 of the ORF exchanged for an F codon. (A) Detection of cells containing replicating virus resulting from RNA transfection. Cells were transfected with equal doses of the in vitro-transcribed RNAs by the DEAE-dextran method and seeded at a sufficient density that 3 days posttransfection a closed layer of cells was obtained. At this time point, cells were fixed and virus-containing cells were visualized by MAb-mediated peroxidase staining. Below the dishes, the NS2-3 cleavage efficiency determined for the respective polyprotein after transient expression is indicated. (B) Analysis of part of the NS2 sequence from RNA obtained at different time points after transfection of RNA derived from pOR/S-F. Cells were split every 3 to 4 days after transfection, and total RNA was prepared from each passage and used as starting material for RT-PCR. RNA from passage 0 was isolated 3 days posttransfection. The amplified fragments were directly sequenced to look for the mutation introduced into pOR/S-F (arrowhead). In lanes C, the sequence of the RT-PCR product derived from the in vitro-transcribed RNA is shown as a control.

Techniques Used: Transfection, Derivative Assay, In Vitro, Staining, Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Mutagenesis

13) Product Images from "Analysis of Endocannabinoid System in Rat Testis During the First Spermatogenetic Wave"

Article Title: Analysis of Endocannabinoid System in Rat Testis During the First Spermatogenetic Wave

Journal: Frontiers in Endocrinology

doi: 10.3389/fendo.2018.00269

Expression of cannabinoid receptors in fetal and post-natal rat testis. Representative fetal testis section from 19.5 days post coitum (dpc) old rats stained with hematoxylin eosin and immunostained for CB1. Arrowheads indicate immunopositive gonocytes. Scale bar: 20 µm (A) . Representative images of RT-PCR analyses showing Cb1 and Actin mRNA expression in rat testes from 1-to-14 days post partum ( dpp ). Cb1 expression was quantified by densitometry analysis, normalized against Actin signals and expressed in OD values (B) . Representative images of RT-PCR analyses showing Cb2 and Actin mRNA expression in rat testes, from 7 to 60 dpp . Cb2 expression was quantified by densitometry analysis, normalized against Actin signals and expressed in OD values (C) . Representative agarose gel image of RT + cDNA (testes from 90 dpp rats), RT− cDNA (testes from 1 to 60 dpp rats) and water (H 2 O) analyzed by PCR using actin primers (D) . All data are reported as the mean ± SEM. Different letters indicate statistically significant differences ( p
Figure Legend Snippet: Expression of cannabinoid receptors in fetal and post-natal rat testis. Representative fetal testis section from 19.5 days post coitum (dpc) old rats stained with hematoxylin eosin and immunostained for CB1. Arrowheads indicate immunopositive gonocytes. Scale bar: 20 µm (A) . Representative images of RT-PCR analyses showing Cb1 and Actin mRNA expression in rat testes from 1-to-14 days post partum ( dpp ). Cb1 expression was quantified by densitometry analysis, normalized against Actin signals and expressed in OD values (B) . Representative images of RT-PCR analyses showing Cb2 and Actin mRNA expression in rat testes, from 7 to 60 dpp . Cb2 expression was quantified by densitometry analysis, normalized against Actin signals and expressed in OD values (C) . Representative agarose gel image of RT + cDNA (testes from 90 dpp rats), RT− cDNA (testes from 1 to 60 dpp rats) and water (H 2 O) analyzed by PCR using actin primers (D) . All data are reported as the mean ± SEM. Different letters indicate statistically significant differences ( p

Techniques Used: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction

RT-PCR analysis of the main AEA and 2-arachidonoylglycerol metabolizing enzymes in rat testis during the first wave of spermatogenesis. Representative images of RT-PCR analyses showing Nape-pld, Faah, Dagl, Magl , and Actin mRNA expression in rat testes from 7 to 60 days post partum ( dpp ) (A) . Representative agarose gel image of RT + cDNA (testes from 90 dpp rats), RT− cDNA (testes from 7 to 60 dpp rats) and water (H 2 O) analyzed by PCR using actin primers (B) . Gene expression was quantified by densitometry analysis and graphed relatively to germ cells appearance or activities (C) . Values for Nape-pld, Faah, Dagl , and Magl signals were normalized against Actin and are expressed as OD values (D) . All data are expressed as the mean ± SEM. Different letters indicate statistically significant differences ( p
Figure Legend Snippet: RT-PCR analysis of the main AEA and 2-arachidonoylglycerol metabolizing enzymes in rat testis during the first wave of spermatogenesis. Representative images of RT-PCR analyses showing Nape-pld, Faah, Dagl, Magl , and Actin mRNA expression in rat testes from 7 to 60 days post partum ( dpp ) (A) . Representative agarose gel image of RT + cDNA (testes from 90 dpp rats), RT− cDNA (testes from 7 to 60 dpp rats) and water (H 2 O) analyzed by PCR using actin primers (B) . Gene expression was quantified by densitometry analysis and graphed relatively to germ cells appearance or activities (C) . Values for Nape-pld, Faah, Dagl , and Magl signals were normalized against Actin and are expressed as OD values (D) . All data are expressed as the mean ± SEM. Different letters indicate statistically significant differences ( p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction

14) Product Images from "Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis ▿ ▿ †"

Article Title: Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis ▿ ▿ †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.05507-11

DNA manipulation and PCR amplification.
Figure Legend Snippet: DNA manipulation and PCR amplification.

Techniques Used: Polymerase Chain Reaction, Amplification

15) Product Images from "Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer"

Article Title: Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkz811

Effect of LT-RPA on stutter peak formation using mono- to tetra-nucleotide repeat microsatellites and CEPH 0144711 cell line. ( A ) Stutter ratio obtained with PCR (20 ng of DNA) and RPA (5 ng DNA, 10.5 mM Mg(OAc) 2 and 40 min incubation at 32°C). ( B ) Microsatellite profiles of the tested mono- to tetra-nucleotide repeat microsatellites using CEPH 0144711 cell line. ( C ) Multiplexing of PCR and low temperature RPA using NR24, HT17 and BAT26 assays and CEPH 0144711 cell line. n (S) and n (L) indicate the small and large allele of a same microsatellite respectively. NRMS, nucleotide repeat microsatellites. All experiments were performed in quadruplicates.
Figure Legend Snippet: Effect of LT-RPA on stutter peak formation using mono- to tetra-nucleotide repeat microsatellites and CEPH 0144711 cell line. ( A ) Stutter ratio obtained with PCR (20 ng of DNA) and RPA (5 ng DNA, 10.5 mM Mg(OAc) 2 and 40 min incubation at 32°C). ( B ) Microsatellite profiles of the tested mono- to tetra-nucleotide repeat microsatellites using CEPH 0144711 cell line. ( C ) Multiplexing of PCR and low temperature RPA using NR24, HT17 and BAT26 assays and CEPH 0144711 cell line. n (S) and n (L) indicate the small and large allele of a same microsatellite respectively. NRMS, nucleotide repeat microsatellites. All experiments were performed in quadruplicates.

Techniques Used: Recombinase Polymerase Amplification, Polymerase Chain Reaction, Incubation, Multiplexing

Effect of the variation of RPA conditions on stutter ratio (SR) compared to PCR using HT17 microsatellite. ( A ) RPA of HT17 using 5 ng of DNA of CEPH 0144711 T17/T17 cell line and 40 min incubation and variable concentrations of Mg(OAc) 2 (5 mM to 14 mM) and of isothermal amplification temperatures (25–42°C). ( B ) PCR amplification of HT17 using 50 pg to 20 ng of DNA of CEPH 0144711 T17/T17 cell line. ( C ) RPA of HT17 using 50 pg to 10 ng of DNA of CEPH 0144711 T17/T17 cell line, 10.5 mM of Mg(OAc) 2 at 32°C during 40 min. ( D ) RPA of HT17 using 5 ng of DNA of CEPH 0144711 T17/T17 cell line, 10.5 mM of Mg(OAc) 2 at 32°C during 10 to 40 min. ( E ) HT17 microsatellite profiles obtained after PCR and RPA (32°C incubation during 40 min with 10.5 mM of Mg(OAc) 2 ) using 20 ng and 5 ng DNA respectively of CEPH cell line harboring the three WT genotypes. Blue dotted lines indicate the HT17 alleles present in the WT cell lines used. Blue and orange peaks correspond to HT17 microsatellite and 140 nucleotide size standard respectively. All experiments were performed in triplicate (B–D), quadruplicate (A) or sextuplicate (B-C, for 50 pg).
Figure Legend Snippet: Effect of the variation of RPA conditions on stutter ratio (SR) compared to PCR using HT17 microsatellite. ( A ) RPA of HT17 using 5 ng of DNA of CEPH 0144711 T17/T17 cell line and 40 min incubation and variable concentrations of Mg(OAc) 2 (5 mM to 14 mM) and of isothermal amplification temperatures (25–42°C). ( B ) PCR amplification of HT17 using 50 pg to 20 ng of DNA of CEPH 0144711 T17/T17 cell line. ( C ) RPA of HT17 using 50 pg to 10 ng of DNA of CEPH 0144711 T17/T17 cell line, 10.5 mM of Mg(OAc) 2 at 32°C during 40 min. ( D ) RPA of HT17 using 5 ng of DNA of CEPH 0144711 T17/T17 cell line, 10.5 mM of Mg(OAc) 2 at 32°C during 10 to 40 min. ( E ) HT17 microsatellite profiles obtained after PCR and RPA (32°C incubation during 40 min with 10.5 mM of Mg(OAc) 2 ) using 20 ng and 5 ng DNA respectively of CEPH cell line harboring the three WT genotypes. Blue dotted lines indicate the HT17 alleles present in the WT cell lines used. Blue and orange peaks correspond to HT17 microsatellite and 140 nucleotide size standard respectively. All experiments were performed in triplicate (B–D), quadruplicate (A) or sextuplicate (B-C, for 50 pg).

Techniques Used: Recombinase Polymerase Amplification, Polymerase Chain Reaction, Incubation, Amplification

Microsatellite profiles of BAT26, D18S61 and D12ATA63 obtained with four blood samples (named B1, B2, B3 and B4) from healthy individuals using PCR (20 ng of DNA in 20 μl) and LT-RPA (5 ng of DNA and 10.5 mM of Mg(OAc) 2 in 10 μl at 32°C). ( A ) Profiles obtained by capillary electrophoresis fragment analysis. ( B ) Profiles obtained by amplicon sequencing. Capillary electrophoresis experiments were performed in duplicates and a representative profile is shown for each duplicated experiment. For amplicon sequencing experiments, the y-axis indicates the read counts for each alleles on a scale of 0–100. NRMS, nucleotide repeat microsatellite.
Figure Legend Snippet: Microsatellite profiles of BAT26, D18S61 and D12ATA63 obtained with four blood samples (named B1, B2, B3 and B4) from healthy individuals using PCR (20 ng of DNA in 20 μl) and LT-RPA (5 ng of DNA and 10.5 mM of Mg(OAc) 2 in 10 μl at 32°C). ( A ) Profiles obtained by capillary electrophoresis fragment analysis. ( B ) Profiles obtained by amplicon sequencing. Capillary electrophoresis experiments were performed in duplicates and a representative profile is shown for each duplicated experiment. For amplicon sequencing experiments, the y-axis indicates the read counts for each alleles on a scale of 0–100. NRMS, nucleotide repeat microsatellite.

Techniques Used: Polymerase Chain Reaction, Recombinase Polymerase Amplification, Electrophoresis, Amplification, Sequencing

HT17 LT-RPA improves the limit of detection of MSI compared to PCR. PCR (20 ng of DNA) and LT-RPA (5 ng of DNA and 10.5 mM of Mg(OAc) 2 at 32°C during 40 min) experiments were conducted on dilution series of cancer cell lines (50%, 25%, 10%, 5% and 1%) harboring T10 to T15 HT17 mutant alleles mixed with T16/T16 WT CEPH cell lines. Only HT17 profiles of the fractions corresponding to the limit of detection of the mutant alleles are shown. Arrows indicate for each cancer cell line used the lowest amount of mutant alleles visible in mutant profiles compared to WT, defining the limit of detection of MSI for each type of mutation. Red and blue dotted lines indicate HT17 genotype of cancer and WT cell lines used respectively. Orange peaks correspond to 140 nucleotide size standard. All experiments were performed in triplicates.
Figure Legend Snippet: HT17 LT-RPA improves the limit of detection of MSI compared to PCR. PCR (20 ng of DNA) and LT-RPA (5 ng of DNA and 10.5 mM of Mg(OAc) 2 at 32°C during 40 min) experiments were conducted on dilution series of cancer cell lines (50%, 25%, 10%, 5% and 1%) harboring T10 to T15 HT17 mutant alleles mixed with T16/T16 WT CEPH cell lines. Only HT17 profiles of the fractions corresponding to the limit of detection of the mutant alleles are shown. Arrows indicate for each cancer cell line used the lowest amount of mutant alleles visible in mutant profiles compared to WT, defining the limit of detection of MSI for each type of mutation. Red and blue dotted lines indicate HT17 genotype of cancer and WT cell lines used respectively. Orange peaks correspond to 140 nucleotide size standard. All experiments were performed in triplicates.

Techniques Used: Recombinase Polymerase Amplification, Polymerase Chain Reaction, Mutagenesis

16) Product Images from "Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT"

Article Title: Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT

Journal: British Journal of Cancer

doi: 10.1038/bjc.2013.168

MRP-1 splicing events observed in ESFT primary tissues. Splicing events observed in primary ESFTs (sample 40) generated by RT-PCR, employing 11 primer mixes spanning the 31 exons of MRP-1 ( Supplementary Figure 1 ). Image shows ethidium bromide-stained amplified products after separation by electrophoresis and visualisation under UV light. *=splice variant; ←=full-length/predicted PCR product; ladder=50 bp DNA ladder;+=RT-positive amplified sample; – =RT-negative control; +H 2 O=control for contamination containing RT, but in which RNA is replaced with H 2 O; H 2 O=negative control in which cDNA is replaced with H 2 O in PCR step.
Figure Legend Snippet: MRP-1 splicing events observed in ESFT primary tissues. Splicing events observed in primary ESFTs (sample 40) generated by RT-PCR, employing 11 primer mixes spanning the 31 exons of MRP-1 ( Supplementary Figure 1 ). Image shows ethidium bromide-stained amplified products after separation by electrophoresis and visualisation under UV light. *=splice variant; ←=full-length/predicted PCR product; ladder=50 bp DNA ladder;+=RT-positive amplified sample; – =RT-negative control; +H 2 O=control for contamination containing RT, but in which RNA is replaced with H 2 O; H 2 O=negative control in which cDNA is replaced with H 2 O in PCR step.

Techniques Used: Generated, Reverse Transcription Polymerase Chain Reaction, Staining, Amplification, Electrophoresis, Variant Assay, Polymerase Chain Reaction, Negative Control

17) Product Images from "HIV Vpr protein upregulates microRNA-122 expression and stimulates hepatitis C virus replication"

Article Title: HIV Vpr protein upregulates microRNA-122 expression and stimulates hepatitis C virus replication

Journal: The Journal of General Virology

doi: 10.1099/vir.0.000169

MiR-122 is essential for the effect of Vpr on HCV 5′ UTR activity, HCV RNA replication and HCV protein expression in the JFH1 infection model and OR6 cell lines. (a) qRT-PCR assay of the expression levels of miR-122 in miR-122-silence Huh7.5.1 cell lines and controls (miR-con). Results were normalized with respect to U6 mRNA. (b) Dual luciferase assay of p5′ UTR-luc in miR-122-silence Huh7.5.1 cell lines transfected with Vpr and controls. (c) Quantitative HCV RNA assay in miR-122-silence Huh7.5.1 cell lines transfected with Vpr and controls. (d) Western blot analysis in miR-122-silence Huh7.5.1 cell lines confirmed the expression of β-actin, NS5A and Vpr. (e) Renilla luciferase assay in miR-122-silence OR6 cell lines transfected with Vpr and controls. (f) Western blot analysis in miR-122-silence OR6 cell lines confirmed the expression of β-actin, NS5A and Vpr. The data are presented as mean ± sd. ** P
Figure Legend Snippet: MiR-122 is essential for the effect of Vpr on HCV 5′ UTR activity, HCV RNA replication and HCV protein expression in the JFH1 infection model and OR6 cell lines. (a) qRT-PCR assay of the expression levels of miR-122 in miR-122-silence Huh7.5.1 cell lines and controls (miR-con). Results were normalized with respect to U6 mRNA. (b) Dual luciferase assay of p5′ UTR-luc in miR-122-silence Huh7.5.1 cell lines transfected with Vpr and controls. (c) Quantitative HCV RNA assay in miR-122-silence Huh7.5.1 cell lines transfected with Vpr and controls. (d) Western blot analysis in miR-122-silence Huh7.5.1 cell lines confirmed the expression of β-actin, NS5A and Vpr. (e) Renilla luciferase assay in miR-122-silence OR6 cell lines transfected with Vpr and controls. (f) Western blot analysis in miR-122-silence OR6 cell lines confirmed the expression of β-actin, NS5A and Vpr. The data are presented as mean ± sd. ** P

Techniques Used: Activity Assay, Expressing, Infection, Quantitative RT-PCR, Luciferase, Transfection, Western Blot

Vpr activates HCV replication in stable Vpr-expressing cells. (a) Quantitative HCV RNA assay in stable vector-Huh7.5.1, Vpr-Huh7.5.1 and Huh7.5.1 cells at 48 h, 72 h and 30 days post-JFH1 infection. (b) Relative fold change of HCV RNA levels. (c) Quantitative PCR for intracellular HCV RNA levels in the stable vector-Huh7.5.1, Vpr-Huh7.5.1 and Huh7.5.1 cells at 30 days post-JFH1 infection. (d, e) Western blotting confirmed the expression of β-actin, NS5A and Vpr in stable Huh7.5.1 cell lines at 72 h (d) and 30 days post-JFH1 infection (e). The data are presented as mean ± sd. ** P
Figure Legend Snippet: Vpr activates HCV replication in stable Vpr-expressing cells. (a) Quantitative HCV RNA assay in stable vector-Huh7.5.1, Vpr-Huh7.5.1 and Huh7.5.1 cells at 48 h, 72 h and 30 days post-JFH1 infection. (b) Relative fold change of HCV RNA levels. (c) Quantitative PCR for intracellular HCV RNA levels in the stable vector-Huh7.5.1, Vpr-Huh7.5.1 and Huh7.5.1 cells at 30 days post-JFH1 infection. (d, e) Western blotting confirmed the expression of β-actin, NS5A and Vpr in stable Huh7.5.1 cell lines at 72 h (d) and 30 days post-JFH1 infection (e). The data are presented as mean ± sd. ** P

Techniques Used: Expressing, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Western Blot

Vpr stimulates HCV replication in JFH1 infection model and OR6 cell lines. (a) Quantitative HCV RNA assay in cell culture supernatants from Huh7.5.1 cells transfected with Vpr and then infected with JFH1 at different times. (b) The relative fold change of HCV RNA levels in cell culture supernatants from Huh7.5.1 cells was measured, with the control non-transfected JFH1-infected Huh7.5.1 cells at 24 h being assigned a value of 1. (c) Quantitative PCR for intracellular HCV RNA levels in the transient Vpr or vector-transfected Huh7.5.1 cells at 72 h post-JFH1 infection. (d) Western blotting confirmed the expression of β-actin, NS5A and Vpr in Huh7.5.1 cell lines infected with JFH1. (e) Renilla luciferase assay in OR6 cell lines transfected with Vpr and controls. (f) Western blot analysis in OR6 cell lines confirmed the expression of β-actin, NS5A and Vpr. The data are presented as mean ± sd. ** P
Figure Legend Snippet: Vpr stimulates HCV replication in JFH1 infection model and OR6 cell lines. (a) Quantitative HCV RNA assay in cell culture supernatants from Huh7.5.1 cells transfected with Vpr and then infected with JFH1 at different times. (b) The relative fold change of HCV RNA levels in cell culture supernatants from Huh7.5.1 cells was measured, with the control non-transfected JFH1-infected Huh7.5.1 cells at 24 h being assigned a value of 1. (c) Quantitative PCR for intracellular HCV RNA levels in the transient Vpr or vector-transfected Huh7.5.1 cells at 72 h post-JFH1 infection. (d) Western blotting confirmed the expression of β-actin, NS5A and Vpr in Huh7.5.1 cell lines infected with JFH1. (e) Renilla luciferase assay in OR6 cell lines transfected with Vpr and controls. (f) Western blot analysis in OR6 cell lines confirmed the expression of β-actin, NS5A and Vpr. The data are presented as mean ± sd. ** P

Techniques Used: Infection, Cell Culture, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation, Western Blot, Expressing, Luciferase

18) Product Images from "A mRNA PCR for the diagnosis of feline infectious peritonitis"

Article Title: A mRNA PCR for the diagnosis of feline infectious peritonitis

Journal: Journal of Virological Methods

doi: 10.1016/j.jviromet.2004.11.012

Specificity controls in FCoV mRNA RT-PCR assay. Lane 1: 100 bp MWM; lane 2: FCoV mRNA positive; lane 3: GAPDH mRNA positive; lane 4: RT-negative control p212; lane 5: mRNA negative PCR control; lane 6: GAPDH negative PCR control; lane 7: 100 bp MWM.
Figure Legend Snippet: Specificity controls in FCoV mRNA RT-PCR assay. Lane 1: 100 bp MWM; lane 2: FCoV mRNA positive; lane 3: GAPDH mRNA positive; lane 4: RT-negative control p212; lane 5: mRNA negative PCR control; lane 6: GAPDH negative PCR control; lane 7: 100 bp MWM.

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

19) Product Images from "Bovipain-2, the falcipain-2 ortholog, is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis"

Article Title: Bovipain-2, the falcipain-2 ortholog, is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis

Journal: Parasites & Vectors

doi: 10.1186/1756-3305-3-113

Transcription of the bovipain-2 gene . Bovipain-2 transcripts were detected by total RNA extraction of in vitro cultured B. bovis merozoites (BboS2P strain), followed by incubation with reverse transcriptase and PCR amplification of the complete ORF (RT+). To rule out DNA contamination, an equal amount of RNA was not incubated with reverse transcriptase and then treated as above (RT-). 1 kb: DNA size standard. The size of the obtained amplicon is marked at the left.
Figure Legend Snippet: Transcription of the bovipain-2 gene . Bovipain-2 transcripts were detected by total RNA extraction of in vitro cultured B. bovis merozoites (BboS2P strain), followed by incubation with reverse transcriptase and PCR amplification of the complete ORF (RT+). To rule out DNA contamination, an equal amount of RNA was not incubated with reverse transcriptase and then treated as above (RT-). 1 kb: DNA size standard. The size of the obtained amplicon is marked at the left.

Techniques Used: RNA Extraction, In Vitro, Cell Culture, Incubation, Polymerase Chain Reaction, Amplification

20) Product Images from "Potential complications when developing gene deletion clones in Xylella fastidiosa"

Article Title: Potential complications when developing gene deletion clones in Xylella fastidiosa

Journal: BMC Research Notes

doi: 10.1186/s13104-015-1117-9

Mixture of wild-type and mutant Xylella fastidiosa strains after second isolation. The XfΔ pilJ mutant confirmed in Figure 3 (isolate 12) was streaked onto periwinkle agar plates amended with kanamycin and the genotype assessed for 32 single colonies. Each number denotes a single colony. A . The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent bands for the transformed XfΔ pilJ mutant strains or the deletion plasmid (P). B . The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔ pilJ strain and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H 2 O, was used as negative controls for each PCR reaction.
Figure Legend Snippet: Mixture of wild-type and mutant Xylella fastidiosa strains after second isolation. The XfΔ pilJ mutant confirmed in Figure 3 (isolate 12) was streaked onto periwinkle agar plates amended with kanamycin and the genotype assessed for 32 single colonies. Each number denotes a single colony. A . The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent bands for the transformed XfΔ pilJ mutant strains or the deletion plasmid (P). B . The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔ pilJ strain and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H 2 O, was used as negative controls for each PCR reaction.

Techniques Used: Mutagenesis, Isolation, Amplification, Transformation Assay, Plasmid Preparation, Positive Control, Polymerase Chain Reaction

Protection of wild-type Xylella fastidiosa from antibiotic selection pressure. Wild-type X. fastidiosa (wt), XfΔ pilJ mutant (J), or an equal mixture of both (M) were grown in PD2 liquid media before being plated onto agar plates with 0, 4, 10, 25, or 50 μg/mL kanamycin, and tested by PCR. The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for wild-type cells and a 2200 bp fragment from the XfΔ pilJ strain. The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for wild-type bacteria and no equivalent bands for the XfΔ pilJ mutant strains. For mixed samples, the AD primers amplified a Xf pilJ strain fragment and the EF primers amplified a wild-type band. The RST31/33 (RST) primers confirmed that the bacteria were X. fastidiosa . Wells of each cell type and kanamycin concentration condition are numbered as follows: (1) AD amplification, (2) EF amplification, and (3) RST amplification.
Figure Legend Snippet: Protection of wild-type Xylella fastidiosa from antibiotic selection pressure. Wild-type X. fastidiosa (wt), XfΔ pilJ mutant (J), or an equal mixture of both (M) were grown in PD2 liquid media before being plated onto agar plates with 0, 4, 10, 25, or 50 μg/mL kanamycin, and tested by PCR. The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for wild-type cells and a 2200 bp fragment from the XfΔ pilJ strain. The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for wild-type bacteria and no equivalent bands for the XfΔ pilJ mutant strains. For mixed samples, the AD primers amplified a Xf pilJ strain fragment and the EF primers amplified a wild-type band. The RST31/33 (RST) primers confirmed that the bacteria were X. fastidiosa . Wells of each cell type and kanamycin concentration condition are numbered as follows: (1) AD amplification, (2) EF amplification, and (3) RST amplification.

Techniques Used: Selection, Mutagenesis, Polymerase Chain Reaction, Amplification, Concentration Assay

Mixture of wild-type and mutant Xylella fastidiosa strains after first isolation. The XfΔ pilJ mutant confirmed in Figure 2 was stored at -80°C, streaked onto periwinkle agar plates amended with kanamycin, and the genotype assessed for 12 single colonies. Each number denotes a single colony. The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed X. fastidiosa and no band for the XfΔ pilJ mutant. Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reaction, while primer reaction without template DNA represented by H 2 O, was used as a negative control.
Figure Legend Snippet: Mixture of wild-type and mutant Xylella fastidiosa strains after first isolation. The XfΔ pilJ mutant confirmed in Figure 2 was stored at -80°C, streaked onto periwinkle agar plates amended with kanamycin, and the genotype assessed for 12 single colonies. Each number denotes a single colony. The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed X. fastidiosa and no band for the XfΔ pilJ mutant. Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reaction, while primer reaction without template DNA represented by H 2 O, was used as a negative control.

Techniques Used: Mutagenesis, Isolation, Amplification, Transformation Assay, Positive Control, Polymerase Chain Reaction, Negative Control

Mixture of wild-type and mutant Xylella fastidiosa strains after third isolation. The XfΔ pilJ mutants confirmed in Figure 4 (isolates 4 and 17) were streaked onto PW agar plates amended with kanamycin and the genotype assessed for 16 single colonies. Each number denotes a single colony. A . The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent band for the XfΔ pilJ strains or the deletion plasmid (P). B . The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔ pilJ strains and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H 2 O, was used as negative controls for both PCR reactions.
Figure Legend Snippet: Mixture of wild-type and mutant Xylella fastidiosa strains after third isolation. The XfΔ pilJ mutants confirmed in Figure 4 (isolates 4 and 17) were streaked onto PW agar plates amended with kanamycin and the genotype assessed for 16 single colonies. Each number denotes a single colony. A . The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent band for the XfΔ pilJ strains or the deletion plasmid (P). B . The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔ pilJ strains and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H 2 O, was used as negative controls for both PCR reactions.

Techniques Used: Mutagenesis, Isolation, Amplification, Transformation Assay, Plasmid Preparation, Positive Control, Polymerase Chain Reaction

Orientation of primers for Xylella fastidiosa pilJ gene deletion. Location of binding sites for PCR primers and length of resulting PCR products for transformed XfΔ pilJ strains and for wild-type control or non-transformed cells. RST31/33 are primers specific to X. fastidiosa and used for bacteria confirmation.
Figure Legend Snippet: Orientation of primers for Xylella fastidiosa pilJ gene deletion. Location of binding sites for PCR primers and length of resulting PCR products for transformed XfΔ pilJ strains and for wild-type control or non-transformed cells. RST31/33 are primers specific to X. fastidiosa and used for bacteria confirmation.

Techniques Used: Binding Assay, Polymerase Chain Reaction, Transformation Assay

21) Product Images from "Identification of New Repetitive Element in Leptospira interrogans Serovar Copenhageni and Its Application to PCR-Based Differentiation of Leptospira Serogroups"

Article Title: Identification of New Repetitive Element in Leptospira interrogans Serovar Copenhageni and Its Application to PCR-Based Differentiation of Leptospira Serogroups

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.39.1.191-195.2001

iRep1 PCR-based molecular typing of reference strains from the genus Leptospira . DNA from boiled culture pellets was used in PCRs with the iRep1 primer for L. interrogans serovar autumnalis strain Akiyami A (lane 2), serovar icterohaemorrhagiae strain RGA (lane 3), serovar copenhageni strain Winjberg (lane 4), serovar wolfii strain 3705 (lane 5), and serovar hardjo strain Hardjoprajitno (lane 6); L. borgpetersenii serovar hardjo strain Lely 607 (lane 7), serovar sejroe strain M84 (lane 8), and serovar castellonis strain Castellon 3 (lane 9); L. weilii serovar celledoni strain Celledoni (lane 10); L. noguchi serovar panama strain CZ 214 K (lane 11); L. santarosai serovar shermani strain 1342 (lane 12); L. kirshneri serovar grippotyphosa strain Moskva V (lane 13); L. wolbachi serovar codice strain CDC (lane 14); L. inadai serovar lyme strain 10 (lane 15); L. fainei serovar hurstbridge strain BUT6 (lane 16); L. meyeri serovar ranarum strain ranae (strain 17); and L. biflexa serovar patoc strain Patoc1 (lane 18). A negative control sample without DNA is shown in lane 19. The positions of the 100-bp size markers are represented in lanes 1 and 20.
Figure Legend Snippet: iRep1 PCR-based molecular typing of reference strains from the genus Leptospira . DNA from boiled culture pellets was used in PCRs with the iRep1 primer for L. interrogans serovar autumnalis strain Akiyami A (lane 2), serovar icterohaemorrhagiae strain RGA (lane 3), serovar copenhageni strain Winjberg (lane 4), serovar wolfii strain 3705 (lane 5), and serovar hardjo strain Hardjoprajitno (lane 6); L. borgpetersenii serovar hardjo strain Lely 607 (lane 7), serovar sejroe strain M84 (lane 8), and serovar castellonis strain Castellon 3 (lane 9); L. weilii serovar celledoni strain Celledoni (lane 10); L. noguchi serovar panama strain CZ 214 K (lane 11); L. santarosai serovar shermani strain 1342 (lane 12); L. kirshneri serovar grippotyphosa strain Moskva V (lane 13); L. wolbachi serovar codice strain CDC (lane 14); L. inadai serovar lyme strain 10 (lane 15); L. fainei serovar hurstbridge strain BUT6 (lane 16); L. meyeri serovar ranarum strain ranae (strain 17); and L. biflexa serovar patoc strain Patoc1 (lane 18). A negative control sample without DNA is shown in lane 19. The positions of the 100-bp size markers are represented in lanes 1 and 20.

Techniques Used: Polymerase Chain Reaction, Negative Control

iRep1 PCR-based molecular typing of human and rat Leptospira strains isolated from an urban epidemic in Salvador, Brazil. DNA from boiled Leptospira culture pellets was amplified with the iRep1 primer. The following samples were identified as L. interrogans serovar copenhageni except for L1-133, shown in lane 3 ( L. interrogans serovar canicola). The following clinical isolates of L. interrogans serovar copenhageni were evaluated: L1-130 (lane 5), L1-212 (lane 6), L8-38 (lane 7), L8-118 (lane 8), and L8-163 (lane 9). Rat isolates included R1-15 (lane 10), R1-98 (lane 11), and R1-147 (lane 12), and R1-152 (lane 13). Lanes 2 and 4 represent reference strains L. interrogans serovar canicola strain Hond Utrecht IV and L. interrogans serovar copenhageni strain Winjberg, respectively. The positions of the 100-bp size markers are represented in lanes 1 and 14.
Figure Legend Snippet: iRep1 PCR-based molecular typing of human and rat Leptospira strains isolated from an urban epidemic in Salvador, Brazil. DNA from boiled Leptospira culture pellets was amplified with the iRep1 primer. The following samples were identified as L. interrogans serovar copenhageni except for L1-133, shown in lane 3 ( L. interrogans serovar canicola). The following clinical isolates of L. interrogans serovar copenhageni were evaluated: L1-130 (lane 5), L1-212 (lane 6), L8-38 (lane 7), L8-118 (lane 8), and L8-163 (lane 9). Rat isolates included R1-15 (lane 10), R1-98 (lane 11), and R1-147 (lane 12), and R1-152 (lane 13). Lanes 2 and 4 represent reference strains L. interrogans serovar canicola strain Hond Utrecht IV and L. interrogans serovar copenhageni strain Winjberg, respectively. The positions of the 100-bp size markers are represented in lanes 1 and 14.

Techniques Used: Polymerase Chain Reaction, Isolation, Amplification

22) Product Images from "Multiple antibiotic resistances among Shiga toxin producing Escherichia coli O157 in feces of dairy cattle farms in Eastern Cape of South Africa"

Article Title: Multiple antibiotic resistances among Shiga toxin producing Escherichia coli O157 in feces of dairy cattle farms in Eastern Cape of South Africa

Journal: BMC Microbiology

doi: 10.1186/s12866-015-0553-y

Agarose gel images of amplicons obtained from PCR with primers designed for str A resistance gene of E. coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are str A (548 bp) gene from O157 strains isolated in this study
Figure Legend Snippet: Agarose gel images of amplicons obtained from PCR with primers designed for str A resistance gene of E. coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are str A (548 bp) gene from O157 strains isolated in this study

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control, Isolation

Gel image of amplicons obtained from multiplex PCR with primers designed for bla - CMY and bla - CXT-M resistance genes of E coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are bla- CMY (507bp) and bla- CXT-M (158 bp) genes from O157 strains isolated in this study
Figure Legend Snippet: Gel image of amplicons obtained from multiplex PCR with primers designed for bla - CMY and bla - CXT-M resistance genes of E coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are bla- CMY (507bp) and bla- CXT-M (158 bp) genes from O157 strains isolated in this study

Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Negative Control, Isolation

Agarose gel image of amplicons obtained from PCR with primers designed for bla - ampC resistance gene of E . coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are bla- ampC (198 bp) gene from O157 strains isolated in this study
Figure Legend Snippet: Agarose gel image of amplicons obtained from PCR with primers designed for bla - ampC resistance gene of E . coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are bla- ampC (198 bp) gene from O157 strains isolated in this study

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control, Isolation

Gel image of amplified PCR products from study isolates with primers designed for stx1 virulent gene. Lane 1 is the MWM (100 bp), lane 2 is the negative control (PCR mix without DNA) with lane 3 as positive control ( E . coli ATCC 35150) while lanes 4 to 13 are stx1 (555 bp) gene amplified from O157 isolates
Figure Legend Snippet: Gel image of amplified PCR products from study isolates with primers designed for stx1 virulent gene. Lane 1 is the MWM (100 bp), lane 2 is the negative control (PCR mix without DNA) with lane 3 as positive control ( E . coli ATCC 35150) while lanes 4 to 13 are stx1 (555 bp) gene amplified from O157 isolates

Techniques Used: Amplification, Polymerase Chain Reaction, Negative Control, Positive Control

23) Product Images from "Direct Amplification and Genotyping of Dientamoeba fragilis from Human Stool Specimens"

Article Title: Direct Amplification and Genotyping of Dientamoeba fragilis from Human Stool Specimens

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.42.2.631-635.2004

Restriction endonuclease digestion of DF1/DF4 PCR products from nine patient samples (lanes 1 to 9) and of PCR products of D. fragilis genotype 1 (lane 11) and genotype 2 (lane 10) with Rsa I (top panel) and Dde I (bottom panel). The size marker (lane M) is a 100-bp ladder.
Figure Legend Snippet: Restriction endonuclease digestion of DF1/DF4 PCR products from nine patient samples (lanes 1 to 9) and of PCR products of D. fragilis genotype 1 (lane 11) and genotype 2 (lane 10) with Rsa I (top panel) and Dde I (bottom panel). The size marker (lane M) is a 100-bp ladder.

Techniques Used: Polymerase Chain Reaction, Marker

24) Product Images from "Analysis of 2-LTR circle junctions of Viral DNA in infected cells"

Article Title: Analysis of 2-LTR circle junctions of Viral DNA in infected cells

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-59745-170-3_6

Quantitation of 2-LTR circle Junction DNA by Real-time PCR. Jurkat cells were infected with WT R3B virus and infected cells DNA was isolated at 2h, 24h and 48h post-infection. Virus infected cell DNA was analyzed for amount of 2-LTR circle junction DNA
Figure Legend Snippet: Quantitation of 2-LTR circle Junction DNA by Real-time PCR. Jurkat cells were infected with WT R3B virus and infected cells DNA was isolated at 2h, 24h and 48h post-infection. Virus infected cell DNA was analyzed for amount of 2-LTR circle junction DNA

Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Infection, Isolation

25) Product Images from "RNA Polymerase II Accumulation in the Promoter-Proximal Region of the Dihydrofolate Reductase and ?-Actin Genes"

Article Title: RNA Polymerase II Accumulation in the Promoter-Proximal Region of the Dihydrofolate Reductase and ?-Actin Genes

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.6.1961-1967.2003

Distribution of RNA Pol II on γ-actin gene. Schematic diagram of the γ-actin gene is shown at the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene. Amplification of an intergenic region served as an internal background control. (A) Specific antibodies used for chromatin IP are shown on top of each gel. Chromatin IP without any antibody or with a nonspecific antibody, Oct2, served as negative controls. PCR analyses of chromatin IP for different regions of the gene are shown. (B) Real-time PCR results are shown for quantification of DNA associated with specific proteins.
Figure Legend Snippet: Distribution of RNA Pol II on γ-actin gene. Schematic diagram of the γ-actin gene is shown at the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene. Amplification of an intergenic region served as an internal background control. (A) Specific antibodies used for chromatin IP are shown on top of each gel. Chromatin IP without any antibody or with a nonspecific antibody, Oct2, served as negative controls. PCR analyses of chromatin IP for different regions of the gene are shown. (B) Real-time PCR results are shown for quantification of DNA associated with specific proteins.

Techniques Used: Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Capping enzyme is concentrated at the promoter of the DHFR gene. HeLa cells containing high numbers of copies of the DHFR gene were cross-linked with formaldehyde. Following sonication, a specific antibody recognizing capping enzyme was added to the chromatin solution for immunoprecipitation of a DNA-capping enzyme complex. PCR was performed to analyze amounts of DNA along the gene that were associated with the capping enzyme. (A) Sonication efficiency of chromatin DNA. The sonicated chromatin solution was analyzed on an ethidium bromide-stained agarose gel after proteinase K treatment and reversal of the cross-links. Two samples are shown, with a 1-kb DNA ladder on the left of the gel. (B) Schematic diagram of the DHFR gene. Black boxes represent exons and thin lines represent introns. DNA fragments for PCR amplification (about 150 nucleotides [nt] long) are depicted as bars under the gene. A pair of primers amplifying an intergenic region downstream of the DHFR gene served as an internal background control. Sequences of primer pairs are presented in Materials and Methods. Numbers below each gel correspond to individual primer sets along the DHFR gene. (C) Chromatin IP with a capping enzyme antibody. PCRs with input DNA (before immunoprecipitation) were performed as controls for amplification efficiency of individual PCR primer sets. Chromatin IP without any antibody and with a nonspecific antibody (Oct2) served as negative controls. Agarose gel analyses of PCR products are shown, with lane numbers corresponding to regions of the gene.
Figure Legend Snippet: Capping enzyme is concentrated at the promoter of the DHFR gene. HeLa cells containing high numbers of copies of the DHFR gene were cross-linked with formaldehyde. Following sonication, a specific antibody recognizing capping enzyme was added to the chromatin solution for immunoprecipitation of a DNA-capping enzyme complex. PCR was performed to analyze amounts of DNA along the gene that were associated with the capping enzyme. (A) Sonication efficiency of chromatin DNA. The sonicated chromatin solution was analyzed on an ethidium bromide-stained agarose gel after proteinase K treatment and reversal of the cross-links. Two samples are shown, with a 1-kb DNA ladder on the left of the gel. (B) Schematic diagram of the DHFR gene. Black boxes represent exons and thin lines represent introns. DNA fragments for PCR amplification (about 150 nucleotides [nt] long) are depicted as bars under the gene. A pair of primers amplifying an intergenic region downstream of the DHFR gene served as an internal background control. Sequences of primer pairs are presented in Materials and Methods. Numbers below each gel correspond to individual primer sets along the DHFR gene. (C) Chromatin IP with a capping enzyme antibody. PCRs with input DNA (before immunoprecipitation) were performed as controls for amplification efficiency of individual PCR primer sets. Chromatin IP without any antibody and with a nonspecific antibody (Oct2) served as negative controls. Agarose gel analyses of PCR products are shown, with lane numbers corresponding to regions of the gene.

Techniques Used: Sonication, Immunoprecipitation, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Amplification, Chromatin Immunoprecipitation

RNA Pol II density determined by nuclear run-on analysis of the DHFR gene. Nuclear run-on analysis was performed as described in Material and Methods. Newly synthesized, 32 P-radiolabeled transcripts were hybridized to PCR products containing specific regions of the DHFR gene. The locations of PCR fragments are diagramed at the top and indicated on the left of the filters. PCR fragments 1 and 3 are genomic DNA, whereas 2 and 4 are cDNA encompassing parts of two adjacent exons. Radiolabeled DHFR RNA was transcribed with SP6 polymerase from each of the four fragments and then pooled and hybridized to the filter as a positive control for hybridization efficiencies among different DHFR probes. Analysis of labeled RNA from the nuclear run-on assay is shown on the right.
Figure Legend Snippet: RNA Pol II density determined by nuclear run-on analysis of the DHFR gene. Nuclear run-on analysis was performed as described in Material and Methods. Newly synthesized, 32 P-radiolabeled transcripts were hybridized to PCR products containing specific regions of the DHFR gene. The locations of PCR fragments are diagramed at the top and indicated on the left of the filters. PCR fragments 1 and 3 are genomic DNA, whereas 2 and 4 are cDNA encompassing parts of two adjacent exons. Radiolabeled DHFR RNA was transcribed with SP6 polymerase from each of the four fragments and then pooled and hybridized to the filter as a positive control for hybridization efficiencies among different DHFR probes. Analysis of labeled RNA from the nuclear run-on assay is shown on the right.

Techniques Used: Synthesized, Polymerase Chain Reaction, Positive Control, Hybridization, Labeling, Nuclear Run-on Assay

26) Product Images from "A Moraxella catarrhalis Two-Component Signal Transduction System Necessary for Growth in Liquid Media Affects Production of Two Lysozyme Inhibitors"

Article Title: A Moraxella catarrhalis Two-Component Signal Transduction System Necessary for Growth in Liquid Media Affects Production of Two Lysozyme Inhibitors

Journal: Infection and Immunity

doi: 10.1128/IAI.02486-14

RNA purification, DNA microarray analysis, and real-time RT-PCR.
Figure Legend Snippet: RNA purification, DNA microarray analysis, and real-time RT-PCR.

Techniques Used: Purification, Microarray, Quantitative RT-PCR

Comparison of DNA microarray and real-time RT-PCR (quantitative RT-PCR [qRT-PCR]) data for selected M. catarrhalis ETSU-9 genes whose expression was affected by inactivation of mesR . (A) Expression levels of 21 selected genes in ETSU-9Δ mesR cells
Figure Legend Snippet: Comparison of DNA microarray and real-time RT-PCR (quantitative RT-PCR [qRT-PCR]) data for selected M. catarrhalis ETSU-9 genes whose expression was affected by inactivation of mesR . (A) Expression levels of 21 selected genes in ETSU-9Δ mesR cells

Techniques Used: Microarray, Quantitative RT-PCR, Expressing

27) Product Images from "Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides"

Article Title: Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

Journal:

doi: 10.1128/AEM.71.6.3025-3032.2005

DNA amplification via PCR.
Figure Legend Snippet: DNA amplification via PCR.

Techniques Used: Amplification, Polymerase Chain Reaction

28) Product Images from "Diet-induced gene expression of isolated pancreatic islets from a polygenic mouse model of the metabolic syndrome"

Article Title: Diet-induced gene expression of isolated pancreatic islets from a polygenic mouse model of the metabolic syndrome

Journal: Diabetologia

doi: 10.1007/s00125-009-1576-4

Enrichment of selected islet genes in LCM samples. mRNA expression genes was determined by quantitative TaqMan RT-PCR for each gene and for Actb from LCM islets (white bars) and total pancreas (black bars) of 8-week-old mice. Expression levels are presented as change in C t or the cycle number of each gene subtracted from the cycle number of beta-actin ( C t value 20.15) for the same cDNA sample. Lower numbers thus correspond to higher expression. Data are means ± SE from five CHF-fed NZL mice. * p ≤ 0.05
Figure Legend Snippet: Enrichment of selected islet genes in LCM samples. mRNA expression genes was determined by quantitative TaqMan RT-PCR for each gene and for Actb from LCM islets (white bars) and total pancreas (black bars) of 8-week-old mice. Expression levels are presented as change in C t or the cycle number of each gene subtracted from the cycle number of beta-actin ( C t value 20.15) for the same cDNA sample. Lower numbers thus correspond to higher expression. Data are means ± SE from five CHF-fed NZL mice. * p ≤ 0.05

Techniques Used: Laser Capture Microdissection, Expressing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay

Islet levels of ChREBP and regulation of lipogenic target genes. Immunohistochemical detection of ChREBP in pancreatic sections from male NZL mice. HF ( a ) and CHF fed ( b ) animals at week 8, with ( c , d ) respective controls with blocking peptide. e TaqMan quantitative PCR validation of ChREBP target genes in islets. Values are normalised to Actb . Data are mean ± SE of five separate mice per group; * p ≤ 0.05. White bars, CHF; black bars, HF; Grey bars, CHF liver; hatched bars, HF liver. Srebp-1 (also known as Srebf1 )
Figure Legend Snippet: Islet levels of ChREBP and regulation of lipogenic target genes. Immunohistochemical detection of ChREBP in pancreatic sections from male NZL mice. HF ( a ) and CHF fed ( b ) animals at week 8, with ( c , d ) respective controls with blocking peptide. e TaqMan quantitative PCR validation of ChREBP target genes in islets. Values are normalised to Actb . Data are mean ± SE of five separate mice per group; * p ≤ 0.05. White bars, CHF; black bars, HF; Grey bars, CHF liver; hatched bars, HF liver. Srebp-1 (also known as Srebf1 )

Techniques Used: Immunohistochemistry, Mouse Assay, Blocking Assay, Real-time Polymerase Chain Reaction

Validation of differential expression in islets by quantitative real-time PCR. TaqMan quantitative PCR validation of genes differentially regulated between CHF (white bars) and HF (black bars). The change in expression is shown for selected genes involved in islet growth and development, protein processing and secretion, metabolism, and signalling. Values are normalised to Actb. Data are mean ± SE of five separate mice per group. * p ≤ 0.05
Figure Legend Snippet: Validation of differential expression in islets by quantitative real-time PCR. TaqMan quantitative PCR validation of genes differentially regulated between CHF (white bars) and HF (black bars). The change in expression is shown for selected genes involved in islet growth and development, protein processing and secretion, metabolism, and signalling. Values are normalised to Actb. Data are mean ± SE of five separate mice per group. * p ≤ 0.05

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

TaqMan quantitative PCR validation of OXPHOS genes in islets. The changes in expression of selected OXPHOS genes are shown. Values are normalised to Actb . Data are mean ± SE of five separate mice per group; * p ≤ 0.05. White bars, CHF; black bars, HF
Figure Legend Snippet: TaqMan quantitative PCR validation of OXPHOS genes in islets. The changes in expression of selected OXPHOS genes are shown. Values are normalised to Actb . Data are mean ± SE of five separate mice per group; * p ≤ 0.05. White bars, CHF; black bars, HF

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

29) Product Images from "Association of Protein Expression and Methylation of DAPK1 with Clinicopathological Features in Invasive Ductal Carcinoma Patients from Kashmir"

Article Title: Association of Protein Expression and Methylation of DAPK1 with Clinicopathological Features in Invasive Ductal Carcinoma Patients from Kashmir

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

doi: 10.31557/APJCP.2019.20.3.839

MSP Analysis of DAPK1 Gene in Breast Cancer Tissues. Existence of PCR products in lane M indicates the presence of methylation. MSP product in lane U indicates the presence of nonmethylation alleles. In vitro SssI methyltransferase–treated and –untreated DNA from normal lymphocytes were used as the positive controls (PC) for methylation and nonmethylation (NC), respectively. L, 100-bp DNA marker ladder; The methylated allele was detected in Cases 1-3, 5, 7 and 8. Unmethylated allele was detected in Cases 4, 6 and 9.
Figure Legend Snippet: MSP Analysis of DAPK1 Gene in Breast Cancer Tissues. Existence of PCR products in lane M indicates the presence of methylation. MSP product in lane U indicates the presence of nonmethylation alleles. In vitro SssI methyltransferase–treated and –untreated DNA from normal lymphocytes were used as the positive controls (PC) for methylation and nonmethylation (NC), respectively. L, 100-bp DNA marker ladder; The methylated allele was detected in Cases 1-3, 5, 7 and 8. Unmethylated allele was detected in Cases 4, 6 and 9.

Techniques Used: Polymerase Chain Reaction, Methylation, In Vitro, Marker

30) Product Images from "Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization"

Article Title: Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035438

Differences in the hybridization kinetics of a target specific reporter probe and a non-target specific reporter probe. A. The diagram shows the kinetics of reporter probe hr-H1-683 in presence of different amounts of hr-H1-683 specific target amplicon. As indicated in the diagram, hybridization speed strongly depends upon the amount of target amplicon present in the reaction. With no target amplicon present in the reaction, reporter probes are available to bind to their complementary capture probes on the array. With increasing amounts of target amplicon, the speed of reporter probe binding to the respective capture probe decreases. Finally, in the presence of approximately the target amplicon concentration expected for a completed PCR- Reaction (150 nM), the reaction speed is at its lowest point. B. The data illustrate that the effect is sequence specific. The speed of hybridization of non-target specific reporter probes to their respective capture probes does not depend on the concentration of target amplicons in solution.
Figure Legend Snippet: Differences in the hybridization kinetics of a target specific reporter probe and a non-target specific reporter probe. A. The diagram shows the kinetics of reporter probe hr-H1-683 in presence of different amounts of hr-H1-683 specific target amplicon. As indicated in the diagram, hybridization speed strongly depends upon the amount of target amplicon present in the reaction. With no target amplicon present in the reaction, reporter probes are available to bind to their complementary capture probes on the array. With increasing amounts of target amplicon, the speed of reporter probe binding to the respective capture probe decreases. Finally, in the presence of approximately the target amplicon concentration expected for a completed PCR- Reaction (150 nM), the reaction speed is at its lowest point. B. The data illustrate that the effect is sequence specific. The speed of hybridization of non-target specific reporter probes to their respective capture probes does not depend on the concentration of target amplicons in solution.

Techniques Used: Hybridization, Amplification, Binding Assay, Concentration Assay, Polymerase Chain Reaction, Sequencing

31) Product Images from "Microdissection of lampbrush chromosomes as an approach for generation of locus-specific FISH-probes and samples for high-throughput sequencing"

Article Title: Microdissection of lampbrush chromosomes as an approach for generation of locus-specific FISH-probes and samples for high-throughput sequencing

Journal: BMC Genomics

doi: 10.1186/s12864-016-2437-4

The scheme illustrating the developed workflow of lampbrush chromosomes (LBC) microdissection. The main steps are as follows: 1. Mechanical microdissection of lampbrush chromosome regions; 2. Primary amplification of isolated DNA material by DOP-PCR. Alternatively, for investigation of RNA-component of chromosomal loci, before the amplification the dissected material was pretreated with DNAse I followed by reverse transcription; 3. FISH-probe generation via reamplification and labeling; 4. Verification of specificity and brightness of the probes by FISH on metaphase chromosomes; 5. High-resolution FISH-mapping of dissected regions on lampbrush chromosomes; 6. Preparation of DNA-libraries for high-throughput sequencing. Processing, visualization and analysis of the sequencing data using web-based bioinformatic platform Galaxy [ 39 ] and genome browser IGV [ 43 , 44 ]. The sequence of actions is depicted by arrows
Figure Legend Snippet: The scheme illustrating the developed workflow of lampbrush chromosomes (LBC) microdissection. The main steps are as follows: 1. Mechanical microdissection of lampbrush chromosome regions; 2. Primary amplification of isolated DNA material by DOP-PCR. Alternatively, for investigation of RNA-component of chromosomal loci, before the amplification the dissected material was pretreated with DNAse I followed by reverse transcription; 3. FISH-probe generation via reamplification and labeling; 4. Verification of specificity and brightness of the probes by FISH on metaphase chromosomes; 5. High-resolution FISH-mapping of dissected regions on lampbrush chromosomes; 6. Preparation of DNA-libraries for high-throughput sequencing. Processing, visualization and analysis of the sequencing data using web-based bioinformatic platform Galaxy [ 39 ] and genome browser IGV [ 43 , 44 ]. The sequence of actions is depicted by arrows

Techniques Used: Laser Capture Microdissection, Amplification, Isolation, Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Labeling, Next-Generation Sequencing, Sequencing

32) Product Images from "Persistent viremia by a novel parvovirus in a slow loris (Nycticebus coucang) with diffuse histiocytic sarcoma"

Article Title: Persistent viremia by a novel parvovirus in a slow loris (Nycticebus coucang) with diffuse histiocytic sarcoma

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2014.00655

Graphical overview of the methods used to detect latent viral forms. (A) Method used to detect episomal genomic DNA. On the top the genome organization of the Sl.L-PV-1, on the bottom a representation of the predicted circular form. Red arrows represent primer binding sites. The use of reverse primer annealing at the beginning of NS1 and forward primers annealing at the end of VP1 allows the amplification the connecting genomic sequence. (B) Restriction enzyme based methods to detect integrated virus. The purple lines represent the host genomic sequence, the orange flashes indicate restriction enzyme sites and the orange boxed indicate artificially ligated adaptors. (B1) It is shown how, after overnight ligation of the digested DNA, the obtained fragment would self-ligate in a circular form and how a specific amplification would allow to amplify the integration site. (B2) It is shown how, after the ligation of specific adaptors to the obtained digested fragments, a semi specific PCR would allow the amplification of the integration site. (C) Method to detect the integrated virus based on repeated Alu sequences in the host genome. Purple boxes represent Alu sequences and red arrows represent primer binding sites. A comparison of the PCR results obtained amplifying the template with either a mixture of Alu binding primers and specific primers or Alu binding primers alone (negative control) would allow to amplify the integration site and determine which amplified fragment on gel corresponds to it.
Figure Legend Snippet: Graphical overview of the methods used to detect latent viral forms. (A) Method used to detect episomal genomic DNA. On the top the genome organization of the Sl.L-PV-1, on the bottom a representation of the predicted circular form. Red arrows represent primer binding sites. The use of reverse primer annealing at the beginning of NS1 and forward primers annealing at the end of VP1 allows the amplification the connecting genomic sequence. (B) Restriction enzyme based methods to detect integrated virus. The purple lines represent the host genomic sequence, the orange flashes indicate restriction enzyme sites and the orange boxed indicate artificially ligated adaptors. (B1) It is shown how, after overnight ligation of the digested DNA, the obtained fragment would self-ligate in a circular form and how a specific amplification would allow to amplify the integration site. (B2) It is shown how, after the ligation of specific adaptors to the obtained digested fragments, a semi specific PCR would allow the amplification of the integration site. (C) Method to detect the integrated virus based on repeated Alu sequences in the host genome. Purple boxes represent Alu sequences and red arrows represent primer binding sites. A comparison of the PCR results obtained amplifying the template with either a mixture of Alu binding primers and specific primers or Alu binding primers alone (negative control) would allow to amplify the integration site and determine which amplified fragment on gel corresponds to it.

Techniques Used: Binding Assay, Amplification, Sequencing, Ligation, Polymerase Chain Reaction, Negative Control

33) Product Images from "TP53 regulates human AlkB homologue 2 expression in glioma resistance to Photofrin-mediated photodynamic therapy"

Article Title: TP53 regulates human AlkB homologue 2 expression in glioma resistance to Photofrin-mediated photodynamic therapy

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6605797

Function of TP53 in DNA repair after Photofrin-mediated PDT. ( A ) Expression of TP53 and p-TP53 at different time points after Ph-PDT. The TP53 mRNA and protein expression were increased at 0.5 and 3.5 h when compared with that of untreated control (CNT) and cells treated with Photofrin alone (DC). p-TP53 levels increased from 3.5 h and reached a maximum at 6.5 h. ( B ) Chromatin immunoprecipitations (ChIPs) were performed using anti-rabbit IgG or anti-p53 antibodies and then analysed by conventional PCR. Input control was DNA sample without any immunoprecipitation. ( C ) ChIP samples analysed and quantified by real-time PCR on two TP53-binding sites at approximately −190 bp and −14 bp from the transcription start site. Data are expressed as mean+s.d. and analysed by using paired t -test of three independent experiments ( *** P
Figure Legend Snippet: Function of TP53 in DNA repair after Photofrin-mediated PDT. ( A ) Expression of TP53 and p-TP53 at different time points after Ph-PDT. The TP53 mRNA and protein expression were increased at 0.5 and 3.5 h when compared with that of untreated control (CNT) and cells treated with Photofrin alone (DC). p-TP53 levels increased from 3.5 h and reached a maximum at 6.5 h. ( B ) Chromatin immunoprecipitations (ChIPs) were performed using anti-rabbit IgG or anti-p53 antibodies and then analysed by conventional PCR. Input control was DNA sample without any immunoprecipitation. ( C ) ChIP samples analysed and quantified by real-time PCR on two TP53-binding sites at approximately −190 bp and −14 bp from the transcription start site. Data are expressed as mean+s.d. and analysed by using paired t -test of three independent experiments ( *** P

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

Photofrin-mediated PDT induces DNA repair gene expression. ( A ) Quantitative real-time RT–PCR of DNA repair genes at 48 h after Photofrin-mediated PDT. ALKBH2 and REV1 were significantly expressed in LD10 and LD 40; DC is used as reference sample and CNT is used to examine the background expression levels of different DNA repair genes in U87 glioma cells. Data are expressed as mean+s.d. and analysed using one-way ANOVA with Tukey's multiple comparison post-test of three independent experiments ( ** P
Figure Legend Snippet: Photofrin-mediated PDT induces DNA repair gene expression. ( A ) Quantitative real-time RT–PCR of DNA repair genes at 48 h after Photofrin-mediated PDT. ALKBH2 and REV1 were significantly expressed in LD10 and LD 40; DC is used as reference sample and CNT is used to examine the background expression levels of different DNA repair genes in U87 glioma cells. Data are expressed as mean+s.d. and analysed using one-way ANOVA with Tukey's multiple comparison post-test of three independent experiments ( ** P

Techniques Used: Expressing, Quantitative RT-PCR

34) Product Images from "Cyclin-dependent Kinase 2-associating Protein 1 Commits Murine Embryonic Stem Cell Differentiation through Retinoblastoma Protein Regulation *"

Article Title: Cyclin-dependent Kinase 2-associating Protein 1 Commits Murine Embryonic Stem Cell Differentiation through Retinoblastoma Protein Regulation *

Journal:

doi: 10.1074/jbc.M109.026088

Gene expression profiles of Cdk2ap1 −/− mESC embryoid bodies. The molecular effects of the deletion of Cdk2ap1 on the regulation of stem cell genes or differentiation-related genes were examined by quantitative reverse transcription-PCR
Figure Legend Snippet: Gene expression profiles of Cdk2ap1 −/− mESC embryoid bodies. The molecular effects of the deletion of Cdk2ap1 on the regulation of stem cell genes or differentiation-related genes were examined by quantitative reverse transcription-PCR

Techniques Used: Expressing, Polymerase Chain Reaction

35) Product Images from "Wide Dispersion and Diversity of Clonally Related Inhibitory Interneurons"

Article Title: Wide Dispersion and Diversity of Clonally Related Inhibitory Interneurons

Journal: Neuron

doi: 10.1016/j.neuron.2015.07.030

Lineage Analysis of Nkx2.1+ Progenitors Using Barcode Retroviral Library (A) Schematic of the QmGFP-OL murine retroviral library. Each retrovirus expresses membrane GFP and contains a 24 bp barcode sequence. (B) EnvA pseudotyped retrovirus libraries were intraventricularly delivered into Nkx2.1-Cre;LSL-Tva embryos at E12.5; brains were harvested and analyzed at P28. (C) Stained neuron outlined for laser capture microdissection and catapulting. (D) Example gel of nested PCR products of dissected cells and GFP negative tissue sections used as controls. (E) Example barcode sequence alignment showing two four-cell clones with matching barcodes. (F) Three dimensional map of two four-cell clones shown in red and blue. Green spheres show cells which did not return a barcode sequence.
Figure Legend Snippet: Lineage Analysis of Nkx2.1+ Progenitors Using Barcode Retroviral Library (A) Schematic of the QmGFP-OL murine retroviral library. Each retrovirus expresses membrane GFP and contains a 24 bp barcode sequence. (B) EnvA pseudotyped retrovirus libraries were intraventricularly delivered into Nkx2.1-Cre;LSL-Tva embryos at E12.5; brains were harvested and analyzed at P28. (C) Stained neuron outlined for laser capture microdissection and catapulting. (D) Example gel of nested PCR products of dissected cells and GFP negative tissue sections used as controls. (E) Example barcode sequence alignment showing two four-cell clones with matching barcodes. (F) Three dimensional map of two four-cell clones shown in red and blue. Green spheres show cells which did not return a barcode sequence.

Techniques Used: Sequencing, Staining, Laser Capture Microdissection, Nested PCR, Clone Assay

36) Product Images from "hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1"

Article Title: hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1

Journal: Nature

doi: 10.1038/nature14280

Autoradiography analysis of the STAU1-RNA complex, and analysis of hybrid reads in a known STAU1 mRNA target, ARF1 a , Autoradiograph of STAU1-RNA complex that was isolated for the hiCLIP experiment. hiCLIP experiments were performed with high and low RNase conditions, and the two controls omitted either the 2nd intermolecular ligation or STAU1 induction. After adaptor ligation, STAU1 cross-linked RNA was radio labeled and the complex was analyzed by denaturing gel electrophoresis and membrane transfer. The size of the band is slightly higher compared to that in Fig. 1a , presumably due to the efficient adaptor ligation that adds to the size of the RNAs (the experiment shown in Fig. 1a didn’t include adaptor ligation). b, Correlation analysis of the non-hybrid read count on each RNA between the replicates of the hiCLIP experiments. c, Schematic representations of ARF1 mRNA and the known STAU1-target RNA duplex, along with the position of STAU1 hybrid reads and cross-link sites identified by non-hybrid reads. The left and right arms of hybrid reads are depicted as black boxes, and lines connect reads originating from the same cDNA. The previously studied STAU1-target RNA duplex 12 , 14 is indicated by green and red boxes. In addition to the known duplex, hybrid reads also identified additional duplexes in the ARF1 3′ UTR. Interestingly, two newly identified duplexes are part of overlapping secondary structures, both of which represent the minimum free energy of folding the local sequence, as predicted by RNAfold 24 (shown on the right). This suggests that some regions of the ARF1 3′ UTR may adopt alternative conformations. The overlapping region of the two structures is shaded in blue. d, The constructs of reporters (ARF1 WT and Δ) used for the validation by formaldehyde crosslinking and co-immunoprecipitation experiment are shown. The reporter has firefly luciferase (FLuc) CDS and ARF1 3′ UTR. e, The ratio of ARF1 WT and Δ in total cell lysate fraction or STAU1 co-immunopreciptated fraction were analyzed by RT-PCR using forward primer annealed to CDS of FLuc and reverse primer annealed to downstream of the deletion site. The ratios (log 2 ) of two populations are compared by Welch’s t test (n = 3). The corresponding Qiaxcel electropherograms are available at: figshare.com/s/5f83e88e929b11e4b77106ec4b8d1f61 .
Figure Legend Snippet: Autoradiography analysis of the STAU1-RNA complex, and analysis of hybrid reads in a known STAU1 mRNA target, ARF1 a , Autoradiograph of STAU1-RNA complex that was isolated for the hiCLIP experiment. hiCLIP experiments were performed with high and low RNase conditions, and the two controls omitted either the 2nd intermolecular ligation or STAU1 induction. After adaptor ligation, STAU1 cross-linked RNA was radio labeled and the complex was analyzed by denaturing gel electrophoresis and membrane transfer. The size of the band is slightly higher compared to that in Fig. 1a , presumably due to the efficient adaptor ligation that adds to the size of the RNAs (the experiment shown in Fig. 1a didn’t include adaptor ligation). b, Correlation analysis of the non-hybrid read count on each RNA between the replicates of the hiCLIP experiments. c, Schematic representations of ARF1 mRNA and the known STAU1-target RNA duplex, along with the position of STAU1 hybrid reads and cross-link sites identified by non-hybrid reads. The left and right arms of hybrid reads are depicted as black boxes, and lines connect reads originating from the same cDNA. The previously studied STAU1-target RNA duplex 12 , 14 is indicated by green and red boxes. In addition to the known duplex, hybrid reads also identified additional duplexes in the ARF1 3′ UTR. Interestingly, two newly identified duplexes are part of overlapping secondary structures, both of which represent the minimum free energy of folding the local sequence, as predicted by RNAfold 24 (shown on the right). This suggests that some regions of the ARF1 3′ UTR may adopt alternative conformations. The overlapping region of the two structures is shaded in blue. d, The constructs of reporters (ARF1 WT and Δ) used for the validation by formaldehyde crosslinking and co-immunoprecipitation experiment are shown. The reporter has firefly luciferase (FLuc) CDS and ARF1 3′ UTR. e, The ratio of ARF1 WT and Δ in total cell lysate fraction or STAU1 co-immunopreciptated fraction were analyzed by RT-PCR using forward primer annealed to CDS of FLuc and reverse primer annealed to downstream of the deletion site. The ratios (log 2 ) of two populations are compared by Welch’s t test (n = 3). The corresponding Qiaxcel electropherograms are available at: figshare.com/s/5f83e88e929b11e4b77106ec4b8d1f61 .

Techniques Used: Autoradiography, Isolation, Ligation, Labeling, Nucleic Acid Electrophoresis, Sequencing, Construct, Immunoprecipitation, Luciferase, Reverse Transcription Polymerase Chain Reaction

37) Product Images from "OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics"

Article Title: OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics

Journal: Journal of Immunology Research

doi: 10.1155/2018/2505818

Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.
Figure Legend Snippet: Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Western Blot, Amplification, Agarose Gel Electrophoresis, Staining, Knock-Out, Expressing

38) Product Images from "A new and simple approach for genotyping Alzheimer's disease presenilin-1 mutant knockin mice"

Article Title: A new and simple approach for genotyping Alzheimer's disease presenilin-1 mutant knockin mice

Journal: Journal of neuroscience methods

doi: 10.1016/j.jneumeth.2009.05.009

Repeatability approaches Two different PCR amplifications were realized from one DNA extraction for the samples 1 to 33. Each PCR product was denaturated and loaded on gels four separate times. (A) Samples 1 to 19 were loaded on two gels migrating in a same electrophoresis (1) as for samples 20 to 33 (electrophoresis 2). (B) Samples 1 to 33 were loaded on two gels (1 to 19 and 20 to 33). Two electrophoresis were realized (3 and 4).
Figure Legend Snippet: Repeatability approaches Two different PCR amplifications were realized from one DNA extraction for the samples 1 to 33. Each PCR product was denaturated and loaded on gels four separate times. (A) Samples 1 to 19 were loaded on two gels migrating in a same electrophoresis (1) as for samples 20 to 33 (electrophoresis 2). (B) Samples 1 to 33 were loaded on two gels (1 to 19 and 20 to 33). Two electrophoresis were realized (3 and 4).

Techniques Used: Polymerase Chain Reaction, DNA Extraction, Electrophoresis

Agarose electrophoresis of PS1 DNA amplified by PCR using the designed primers The specific set of primers designed for PS1 gene generated a 231 bp PCR product in three different DNA samples tested whose genotypes were determined using the classical method (+/+, +/KI, KI/KI). PCR was performed in triplicate for each sample. No PCR product was detected in the negative control lane (T−).
Figure Legend Snippet: Agarose electrophoresis of PS1 DNA amplified by PCR using the designed primers The specific set of primers designed for PS1 gene generated a 231 bp PCR product in three different DNA samples tested whose genotypes were determined using the classical method (+/+, +/KI, KI/KI). PCR was performed in triplicate for each sample. No PCR product was detected in the negative control lane (T−).

Techniques Used: Electrophoresis, Amplification, Polymerase Chain Reaction, Generated, Negative Control

39) Product Images from "High expression of DDR1 is associated with the poor prognosis in Chinese patients with pancreatic ductal adenocarcinoma"

Article Title: High expression of DDR1 is associated with the poor prognosis in Chinese patients with pancreatic ductal adenocarcinoma

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-015-0202-1

DDR1 expression is increased in PDAC at mRNA level. a increased DDR1 mRNA expression in 30 matched tumor (T) and non-tumor tissue (N) was detected by real-time quantitative PCR. b DDR1 expression in Buchholz pancreas grouped by normal pancreatic duct (1) and PDAC (2)
Figure Legend Snippet: DDR1 expression is increased in PDAC at mRNA level. a increased DDR1 mRNA expression in 30 matched tumor (T) and non-tumor tissue (N) was detected by real-time quantitative PCR. b DDR1 expression in Buchholz pancreas grouped by normal pancreatic duct (1) and PDAC (2)

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

40) Product Images from "rpoB Gene Analysis as a Novel Strategy for Identification of Spirochetes from the Genera Borrelia, Treponema, and Leptospira"

Article Title: rpoB Gene Analysis as a Novel Strategy for Identification of Spirochetes from the Genera Borrelia, Treponema, and Leptospira

Journal: Journal of Clinical Microbiology

doi:

Specific PCR amplification of the spirochetal rpoB gene. The PCR assay was performed by using Taq polymerase with an annealing temperature of 52°C. Lanes: 1, molecular mass markers (DNA molecular weight marker VI; Boehringer); 2, Borrelia burgdorferi ; 3, Borrelia recurrentis ; 4, Treponema pallidum ; 5, Leptospira biflexa serovar patoc; 6, Leptospira interrogans , serovar australis; 7, Leptospira interrogans , serovar icterohaemmorragiae; 8, Escherichia coli ; 9, Staphylococcus aureus ; 10, Streptococcus salivarius ; 11, Pseudomonas aeruginosa ; 12, negative control without DNA. (A) rpoB gene primers LTB 1730F and LTB 2900R. (B) rpoB gene primers LTB 1730F and LTB 3700R. (C) 16S rRNA gene primers FD1 and RP2.
Figure Legend Snippet: Specific PCR amplification of the spirochetal rpoB gene. The PCR assay was performed by using Taq polymerase with an annealing temperature of 52°C. Lanes: 1, molecular mass markers (DNA molecular weight marker VI; Boehringer); 2, Borrelia burgdorferi ; 3, Borrelia recurrentis ; 4, Treponema pallidum ; 5, Leptospira biflexa serovar patoc; 6, Leptospira interrogans , serovar australis; 7, Leptospira interrogans , serovar icterohaemmorragiae; 8, Escherichia coli ; 9, Staphylococcus aureus ; 10, Streptococcus salivarius ; 11, Pseudomonas aeruginosa ; 12, negative control without DNA. (A) rpoB gene primers LTB 1730F and LTB 2900R. (B) rpoB gene primers LTB 1730F and LTB 3700R. (C) 16S rRNA gene primers FD1 and RP2.

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

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Amplification:

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Article Snippet: .. PCR amplification was conducted using 100 ng of gemonic DNA in a total volume of 20 μl with forward and reverse primers (400 nM each) using DreamTaq PCR Master Mix (Thermo Scientific). .. At the first round, fragments 1–3 were amplified by PCR with primer sets of CA5F and CA3R, CA1F and CA2R, CA4F and CA5R, respectively.

Article Title: Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes *
Article Snippet: .. For in vitro cleavage kinetics, ribozyme constructs were either amplified from their respective genomes or synthesized by mutual priming using primers listed below and the DreamTaq PCR master mix (Fermentas). .. The constructs were designed to include a 40-nucleotide leader sequence, the full ribozyme, and a short (2–8 nucleotide) tail.

Article Title: Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate
Article Snippet: .. T-RFLP Monitoring Amplification of a 900 bp fragment of the 16S rRNA gene for T-RFLP analysis from DNA extracts was achieved using DreamTaq PCR Master Mix (Thermo Fisher) and a Cy5-labeled 8F forward primer ( ) and 907R ( ) as described previously ( ). .. Up to 4 μL of PCR product were digested in a 10 μL reaction with 1 U of the restriction endonuclease HaeIII (Thermo Scientific) and 1 μL appropriate buffer.

In Vitro:

Article Title: Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes *
Article Snippet: .. For in vitro cleavage kinetics, ribozyme constructs were either amplified from their respective genomes or synthesized by mutual priming using primers listed below and the DreamTaq PCR master mix (Fermentas). .. The constructs were designed to include a 40-nucleotide leader sequence, the full ribozyme, and a short (2–8 nucleotide) tail.

Synthesized:

Article Title: Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes *
Article Snippet: .. For in vitro cleavage kinetics, ribozyme constructs were either amplified from their respective genomes or synthesized by mutual priming using primers listed below and the DreamTaq PCR master mix (Fermentas). .. The constructs were designed to include a 40-nucleotide leader sequence, the full ribozyme, and a short (2–8 nucleotide) tail.

Construct:

Article Title: Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes *
Article Snippet: .. For in vitro cleavage kinetics, ribozyme constructs were either amplified from their respective genomes or synthesized by mutual priming using primers listed below and the DreamTaq PCR master mix (Fermentas). .. The constructs were designed to include a 40-nucleotide leader sequence, the full ribozyme, and a short (2–8 nucleotide) tail.

Terminal Restriction Fragment Length Polymorphism:

Article Title: Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate
Article Snippet: .. T-RFLP Monitoring Amplification of a 900 bp fragment of the 16S rRNA gene for T-RFLP analysis from DNA extracts was achieved using DreamTaq PCR Master Mix (Thermo Fisher) and a Cy5-labeled 8F forward primer ( ) and 907R ( ) as described previously ( ). .. Up to 4 μL of PCR product were digested in a 10 μL reaction with 1 U of the restriction endonuclease HaeIII (Thermo Scientific) and 1 μL appropriate buffer.

Polymerase Chain Reaction:

Article Title: Using an in-vitro biofilm model to assess the virulence potential of Bacterial Vaginosis or non-Bacterial Vaginosis Gardnerella vaginalis isolates
Article Snippet: .. Genomic DNA was extracted as described before and the thermocycling program (Mini-MJ, Bio-Rad, Hercules, CA, USA) was performed using the DreamTaq PCR Master Mix 2x (Finnzymes, Espoo, Finland) and consisted on the following steps: 94 °C for 2 minutes followed by 40 cycles of 94 °C for 30 seconds, 58 °C for 30 seconds, 72 °C for 60 seconds and finally 72 °C for 5 minutes. ..

Article Title: The Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species
Article Snippet: .. PCR amplification was conducted using 100 ng of gemonic DNA in a total volume of 20 μl with forward and reverse primers (400 nM each) using DreamTaq PCR Master Mix (Thermo Scientific). .. At the first round, fragments 1–3 were amplified by PCR with primer sets of CA5F and CA3R, CA1F and CA2R, CA4F and CA5R, respectively.

Article Title: Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes *
Article Snippet: .. For in vitro cleavage kinetics, ribozyme constructs were either amplified from their respective genomes or synthesized by mutual priming using primers listed below and the DreamTaq PCR master mix (Fermentas). .. The constructs were designed to include a 40-nucleotide leader sequence, the full ribozyme, and a short (2–8 nucleotide) tail.

Article Title: Phenotype-Independent Isolation of Interspecies Saccharomyces Hybrids by Dual-Dye Fluorescent Staining and Fluorescence-Activated Cell Sorting
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Article Snippet: .. Briefly, each 50-μl PCR mixture contained 1 μl of template, 25 μl of DreamTaq PCR master mix (MBI Fermentas), 2 μl of each primer (10 μM), and 20 μl of Milli-Q water. ..

Article Title: Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate
Article Snippet: .. T-RFLP Monitoring Amplification of a 900 bp fragment of the 16S rRNA gene for T-RFLP analysis from DNA extracts was achieved using DreamTaq PCR Master Mix (Thermo Fisher) and a Cy5-labeled 8F forward primer ( ) and 907R ( ) as described previously ( ). .. Up to 4 μL of PCR product were digested in a 10 μL reaction with 1 U of the restriction endonuclease HaeIII (Thermo Scientific) and 1 μL appropriate buffer.

Article Title: Molecular prevalence and haemato-biochemical profile of canine monocytic ehrlichiosis in dogs in and around Hisar, Haryana, India
Article Snippet: .. The reaction (10 μl) contained 1.0 μl of template DNA in DreamTaq PCR master mix (Thermo Fisher Scientific) containing 5.0 μl DreamTaq polymerase, optimized DreamTaq buffer, MgCl2 and dNTPs; 0.3 μl of each primer and DMSO and 3.1 μl NFW. .. The thermocycling conditions consisted of initial denaturation at 94 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 45 s and extension at 72 °C for 30 s. This was followed by a final extension at 72 °C for 6 min. All the PCR products and a molecular weight marker (100 base-pair ladder) were electrophoresed through 1.5% agarose gels stained with ethidium bromide in Tris-acetate-EDTA (TAE) buffer for 35 min at 80 V and the DNA fragments were visualized under UV fluorescence using a gel documentation system (GeNei Uvitec, UK).

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  • 99
    Thermo Fisher dreamtaq green pcr master mix 2x
    BFR1 deletion does not affect PMT1 and PMT2 transcript localization. ( A ) and ( B ): JCY017 (wild type BFR1-3xHA) cells were grown in YPD medium, lysed and total cell extracts were subjected to one step ultracentrifugation. ( A ) Western blot analysis of total cell lysates (TCL), soluble and membrane fractions (SF and MF respectively) upon one step ultracentrifugation. Equivalents to 0.25 OD 600 were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 served as a membrane marker and G6PDH as a cytosolic marker. Bfr1-3xHA was detected using the HA-tag. ( B ) <t>RT-PCR</t> analysis of PMT2 mRNA from soluble and membrane fractions upon one step ultracentrifugation. Total RNA was extracted from respective fractions and cDNA was prepared. PMT2 mRNA from each fraction was normalized to ACT1 mRNA. Results show the average membrane to soluble PMT2 mRNA ratio from three independent experiments and error bars represent the confidence interval. For statistical significance one-sample t -test was performed on log2 −ΔΔCt . ( C ), ( D ), and ( E ): JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells were grown in YPD medium, lysed and total cell extracts were subjected to sucrose step gradient centrifugation. ( C ) Absorbance 260 profile of fractions collected upon sucrose step gradient centrifugation (upper panel) and agarose gel electrophoresis of equivalent amounts of each fraction (lower panel). F5, F10, and F16 indicate fractions selected for further analysis. Black and grey arrows next to the agarose gel depict ribosomal subunits 60S and 40S. ( D ) Western blot analysis of total cell lysates (TCL) and selected sucrose gradient fractions from JCY017 (wild type BFR1-3xHA) cells. 0.25 OD 600 units of total cell extract and equivalents of selected fractions were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 and G6PDH were detected exclusively in fractions F16 and F5 respectively confirming successful fractionation. The ribosomal protein Rpl5 was mainly detected in fractions F10 and F16 that represent cytoplasmic and membrane bound polysomes respectively. The weaker Rpl5 signal detected in fraction F5 probably emanates from free cytosolic ribosomes. ( E ) Semi-quantitative PCR analysis of PMT1 and PMT2 mRNA from sucrose gradient fractions F10 and F16 from JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells. Total RNA was extracted from respective fractions, cDNA was prepared and a 1:20 dilution was used as template in a standard <t>DreamTaq</t> PCR reaction. ACT1 that served as a loading control also shows strong engagement in the ER membrane containing fraction F16 in line with reports that the ER is a general translation hub even for cytosolic proteins [ 53 ]. Results are representative of two independent fractionations. N.s. = not significant.
    Dreamtaq Green Pcr Master Mix 2x, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dreamtaq green pcr master mix 2x/product/Thermo Fisher
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    Thermo Fisher sybr green pcr master mix
    Silencing of Snail in Caki-2 by a lentivirus-delivered short inhibitory RNA. A. Map of the lentivirualRNAi vectors [pCMV-G NR-U6-shRNA]. U6, polymerase III promoter to drive the transcription of shRNAs; GFP (green fluorescent protein) were driven by CMV promoter. shRNAs were inserted into HindIII and BamHI sites. B. Strategy plan for generating Snail shRNA constructs. pCMV-G NR-U6 vector was linearized by HindIII and BamH1, and then designed shRNAs were inserted into this site. The positive colonies were screened by colony <t>PCR</t> using the primers flanking the inserted site of shRNA. Selected Colonies were finally confirmed by sequencing. C. Quantitative real-time RT-PCR compare the knock-down efficiency of 3 Snail shRNAs in Caki-2 cells. RNAs were isolated from cells and gene reverse transcription was performed. Quantitative PCR was performed by using <t>SYBR</t> Green PCR Master Mix. Data were normalized to GAPDH mRNA levels, by using efficiency (2 -ΔΔCt ) method. The data were analyzed by student t -test, and were presented as the means ± s.e.m (n = 3). D. Western blotting assay confirmed that effectiveness of Snail shRNA viruses on protein level.The intensity of bands were determined by Image J, and ratio of Snail to GAPDH were shown here.
    Sybr Green Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12218 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qrt pcr reaction mixture
    Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by <t>qRT-PCR</t> with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P
    Qrt Pcr Reaction Mixture, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BFR1 deletion does not affect PMT1 and PMT2 transcript localization. ( A ) and ( B ): JCY017 (wild type BFR1-3xHA) cells were grown in YPD medium, lysed and total cell extracts were subjected to one step ultracentrifugation. ( A ) Western blot analysis of total cell lysates (TCL), soluble and membrane fractions (SF and MF respectively) upon one step ultracentrifugation. Equivalents to 0.25 OD 600 were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 served as a membrane marker and G6PDH as a cytosolic marker. Bfr1-3xHA was detected using the HA-tag. ( B ) RT-PCR analysis of PMT2 mRNA from soluble and membrane fractions upon one step ultracentrifugation. Total RNA was extracted from respective fractions and cDNA was prepared. PMT2 mRNA from each fraction was normalized to ACT1 mRNA. Results show the average membrane to soluble PMT2 mRNA ratio from three independent experiments and error bars represent the confidence interval. For statistical significance one-sample t -test was performed on log2 −ΔΔCt . ( C ), ( D ), and ( E ): JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells were grown in YPD medium, lysed and total cell extracts were subjected to sucrose step gradient centrifugation. ( C ) Absorbance 260 profile of fractions collected upon sucrose step gradient centrifugation (upper panel) and agarose gel electrophoresis of equivalent amounts of each fraction (lower panel). F5, F10, and F16 indicate fractions selected for further analysis. Black and grey arrows next to the agarose gel depict ribosomal subunits 60S and 40S. ( D ) Western blot analysis of total cell lysates (TCL) and selected sucrose gradient fractions from JCY017 (wild type BFR1-3xHA) cells. 0.25 OD 600 units of total cell extract and equivalents of selected fractions were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 and G6PDH were detected exclusively in fractions F16 and F5 respectively confirming successful fractionation. The ribosomal protein Rpl5 was mainly detected in fractions F10 and F16 that represent cytoplasmic and membrane bound polysomes respectively. The weaker Rpl5 signal detected in fraction F5 probably emanates from free cytosolic ribosomes. ( E ) Semi-quantitative PCR analysis of PMT1 and PMT2 mRNA from sucrose gradient fractions F10 and F16 from JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells. Total RNA was extracted from respective fractions, cDNA was prepared and a 1:20 dilution was used as template in a standard DreamTaq PCR reaction. ACT1 that served as a loading control also shows strong engagement in the ER membrane containing fraction F16 in line with reports that the ER is a general translation hub even for cytosolic proteins [ 53 ]. Results are representative of two independent fractionations. N.s. = not significant.

    Journal: International Journal of Molecular Sciences

    Article Title: Translational Regulation of Pmt1 and Pmt2 by Bfr1 Affects Unfolded Protein O-Mannosylation

    doi: 10.3390/ijms20246220

    Figure Lengend Snippet: BFR1 deletion does not affect PMT1 and PMT2 transcript localization. ( A ) and ( B ): JCY017 (wild type BFR1-3xHA) cells were grown in YPD medium, lysed and total cell extracts were subjected to one step ultracentrifugation. ( A ) Western blot analysis of total cell lysates (TCL), soluble and membrane fractions (SF and MF respectively) upon one step ultracentrifugation. Equivalents to 0.25 OD 600 were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 served as a membrane marker and G6PDH as a cytosolic marker. Bfr1-3xHA was detected using the HA-tag. ( B ) RT-PCR analysis of PMT2 mRNA from soluble and membrane fractions upon one step ultracentrifugation. Total RNA was extracted from respective fractions and cDNA was prepared. PMT2 mRNA from each fraction was normalized to ACT1 mRNA. Results show the average membrane to soluble PMT2 mRNA ratio from three independent experiments and error bars represent the confidence interval. For statistical significance one-sample t -test was performed on log2 −ΔΔCt . ( C ), ( D ), and ( E ): JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells were grown in YPD medium, lysed and total cell extracts were subjected to sucrose step gradient centrifugation. ( C ) Absorbance 260 profile of fractions collected upon sucrose step gradient centrifugation (upper panel) and agarose gel electrophoresis of equivalent amounts of each fraction (lower panel). F5, F10, and F16 indicate fractions selected for further analysis. Black and grey arrows next to the agarose gel depict ribosomal subunits 60S and 40S. ( D ) Western blot analysis of total cell lysates (TCL) and selected sucrose gradient fractions from JCY017 (wild type BFR1-3xHA) cells. 0.25 OD 600 units of total cell extract and equivalents of selected fractions were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 and G6PDH were detected exclusively in fractions F16 and F5 respectively confirming successful fractionation. The ribosomal protein Rpl5 was mainly detected in fractions F10 and F16 that represent cytoplasmic and membrane bound polysomes respectively. The weaker Rpl5 signal detected in fraction F5 probably emanates from free cytosolic ribosomes. ( E ) Semi-quantitative PCR analysis of PMT1 and PMT2 mRNA from sucrose gradient fractions F10 and F16 from JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells. Total RNA was extracted from respective fractions, cDNA was prepared and a 1:20 dilution was used as template in a standard DreamTaq PCR reaction. ACT1 that served as a loading control also shows strong engagement in the ER membrane containing fraction F16 in line with reports that the ER is a general translation hub even for cytosolic proteins [ 53 ]. Results are representative of two independent fractionations. N.s. = not significant.

    Article Snippet: Semi-quantitative PCR was performed using the DreamTaq Green PCR master mix (#K1081, Thermo Fisher Scientific; Waltham, MA, USA) according to manufacturer´s instructions.

    Techniques: Western Blot, Marker, Reverse Transcription Polymerase Chain Reaction, Gradient Centrifugation, Agarose Gel Electrophoresis, Fractionation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Silencing of Snail in Caki-2 by a lentivirus-delivered short inhibitory RNA. A. Map of the lentivirualRNAi vectors [pCMV-G NR-U6-shRNA]. U6, polymerase III promoter to drive the transcription of shRNAs; GFP (green fluorescent protein) were driven by CMV promoter. shRNAs were inserted into HindIII and BamHI sites. B. Strategy plan for generating Snail shRNA constructs. pCMV-G NR-U6 vector was linearized by HindIII and BamH1, and then designed shRNAs were inserted into this site. The positive colonies were screened by colony PCR using the primers flanking the inserted site of shRNA. Selected Colonies were finally confirmed by sequencing. C. Quantitative real-time RT-PCR compare the knock-down efficiency of 3 Snail shRNAs in Caki-2 cells. RNAs were isolated from cells and gene reverse transcription was performed. Quantitative PCR was performed by using SYBR Green PCR Master Mix. Data were normalized to GAPDH mRNA levels, by using efficiency (2 -ΔΔCt ) method. The data were analyzed by student t -test, and were presented as the means ± s.e.m (n = 3). D. Western blotting assay confirmed that effectiveness of Snail shRNA viruses on protein level.The intensity of bands were determined by Image J, and ratio of Snail to GAPDH were shown here.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Synergistic effects of snail and quercetin on renal cell carcinoma Caki-2 by altering AKT/mTOR/ERK1/2 signaling pathways

    doi:

    Figure Lengend Snippet: Silencing of Snail in Caki-2 by a lentivirus-delivered short inhibitory RNA. A. Map of the lentivirualRNAi vectors [pCMV-G NR-U6-shRNA]. U6, polymerase III promoter to drive the transcription of shRNAs; GFP (green fluorescent protein) were driven by CMV promoter. shRNAs were inserted into HindIII and BamHI sites. B. Strategy plan for generating Snail shRNA constructs. pCMV-G NR-U6 vector was linearized by HindIII and BamH1, and then designed shRNAs were inserted into this site. The positive colonies were screened by colony PCR using the primers flanking the inserted site of shRNA. Selected Colonies were finally confirmed by sequencing. C. Quantitative real-time RT-PCR compare the knock-down efficiency of 3 Snail shRNAs in Caki-2 cells. RNAs were isolated from cells and gene reverse transcription was performed. Quantitative PCR was performed by using SYBR Green PCR Master Mix. Data were normalized to GAPDH mRNA levels, by using efficiency (2 -ΔΔCt ) method. The data were analyzed by student t -test, and were presented as the means ± s.e.m (n = 3). D. Western blotting assay confirmed that effectiveness of Snail shRNA viruses on protein level.The intensity of bands were determined by Image J, and ratio of Snail to GAPDH were shown here.

    Article Snippet: Quantitative PCR was performed with SYBR Green PCR Master Mix (Thermo, F-415XL) on Applied Biosystems 7300 Fast Real-Time PCR System.

    Techniques: shRNA, Construct, Plasmid Preparation, Polymerase Chain Reaction, Sequencing, Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Western Blot

    Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Over Expression, Expressing, Plasmid Preparation, Construct, Quantitative RT-PCR, High Performance Liquid Chromatography

    Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR

    PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR