pcr buffer  (Roche)


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    Structured Review

    Roche pcr buffer
    Human papillomavirus (HPV)16 type-specific polymerase chain reaction products. Lane 4: 50 bp ladder; lanes 3, 5-8: <t>HPV16</t> (+) ve samples showing specific band size at 116 bp; lane 9: HPV16 (–)ve sample; lane 2: Positive control; lane 1: Negative control
    Pcr Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India"

    Article Title: Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India

    Journal: Journal of Mid-Life Health

    doi: 10.4103/0976-7800.127786

    Human papillomavirus (HPV)16 type-specific polymerase chain reaction products. Lane 4: 50 bp ladder; lanes 3, 5-8: HPV16 (+) ve samples showing specific band size at 116 bp; lane 9: HPV16 (–)ve sample; lane 2: Positive control; lane 1: Negative control
    Figure Legend Snippet: Human papillomavirus (HPV)16 type-specific polymerase chain reaction products. Lane 4: 50 bp ladder; lanes 3, 5-8: HPV16 (+) ve samples showing specific band size at 116 bp; lane 9: HPV16 (–)ve sample; lane 2: Positive control; lane 1: Negative control

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

    Flow chart depicting study design. TS-PCR: Type-specific polymerase chain reaction, HPV: Human papillomavirus, H and E: Hematoxylin and eosin, PAS: Periodic Acid-Schiff
    Figure Legend Snippet: Flow chart depicting study design. TS-PCR: Type-specific polymerase chain reaction, HPV: Human papillomavirus, H and E: Hematoxylin and eosin, PAS: Periodic Acid-Schiff

    Techniques Used: Flow Cytometry, Polymerase Chain Reaction

    2) Product Images from "Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters"

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.6.3588-3592.2004

    The linear range of the real-time PCR method when detecting purified DNA from C. jejuni CCUG 11284 on the RotorGene 3000 (5 × 10 1 to 1 × 10 7 copies; □) and the ABI-PRISM 7700 (10 3 to 10 7 copies; ▴). A real-time PCR sample
    Figure Legend Snippet: The linear range of the real-time PCR method when detecting purified DNA from C. jejuni CCUG 11284 on the RotorGene 3000 (5 × 10 1 to 1 × 10 7 copies; □) and the ABI-PRISM 7700 (10 3 to 10 7 copies; ▴). A real-time PCR sample

    Techniques Used: Real-time Polymerase Chain Reaction, Purification

    3) Product Images from "Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents"

    Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-018-0594-9

    Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)
    Figure Legend Snippet: Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

    Techniques Used: Methylation, Mass Spectrometry, Polymerase Chain Reaction, Derivative Assay, Positive Control, Molecular Weight

    4) Product Images from "Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori"

    Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

    Journal: Iranian Journal of Basic Medical Sciences

    doi:

    Antigenic region of the VacA gene from Helicobacter pylori amplified by PCR. Lane 1: DNA marker, Lane 2: PCR product
    Figure Legend Snippet: Antigenic region of the VacA gene from Helicobacter pylori amplified by PCR. Lane 1: DNA marker, Lane 2: PCR product

    Techniques Used: Amplification, Polymerase Chain Reaction, Marker

    5) Product Images from "Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1"

    Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1

    Journal: Translational Oncology

    doi:

    Galanin methylation analysis using the MSP assay and galanin expression by quantitative RT-PCR in HNSCC tumor samples. (A) Representative examples of MSP of galanin in primary tumors from Hamamatsu University Hospital, showing samples that are methylated
    Figure Legend Snippet: Galanin methylation analysis using the MSP assay and galanin expression by quantitative RT-PCR in HNSCC tumor samples. (A) Representative examples of MSP of galanin in primary tumors from Hamamatsu University Hospital, showing samples that are methylated

    Techniques Used: Methylation, MSP Assay, Expressing, Quantitative RT-PCR

    6) Product Images from "Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor"

    Article Title: Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.14475

    HPV Nested PCR. The presence of HPV in the OSCC samples were tested using (a) HPV primer PGMY09/11 and (b) nested HPV primer GP5 + /6 + . A representative gel is shown from 84 samples tested with the two consensus primers displaying positive PCR amplification for PGMY09/11 (450bp) and nested GP5 + /6 + (150bp). Positive Control (PC) using DNA from HeLa cells and negative control (NC) were included in the experiments. Marker and sample ID are indicated.
    Figure Legend Snippet: HPV Nested PCR. The presence of HPV in the OSCC samples were tested using (a) HPV primer PGMY09/11 and (b) nested HPV primer GP5 + /6 + . A representative gel is shown from 84 samples tested with the two consensus primers displaying positive PCR amplification for PGMY09/11 (450bp) and nested GP5 + /6 + (150bp). Positive Control (PC) using DNA from HeLa cells and negative control (NC) were included in the experiments. Marker and sample ID are indicated.

    Techniques Used: Nested PCR, Polymerase Chain Reaction, Amplification, Positive Control, Negative Control, Marker

    7) Product Images from "CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1"

    Article Title: CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi:

    CD8+ cells express IL-17 within the allografts of T-bet-/- recipients. A , Intragraft expression of IL-17 was assessed by real-time RT-PCR. From top to bottom, RNA was harvested from allografts transplanted into the following mice: unmodified WT, unmodified
    Figure Legend Snippet: CD8+ cells express IL-17 within the allografts of T-bet-/- recipients. A , Intragraft expression of IL-17 was assessed by real-time RT-PCR. From top to bottom, RNA was harvested from allografts transplanted into the following mice: unmodified WT, unmodified

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay

    8) Product Images from "DNA Damage, Homology-Directed Repair, and DNA Methylation"

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030110

    Dnmt1 Inhibits the Expression of Recombinant GFP Genes Wild-type or Dnmt1−/− ES cells carrying DR-GFP were transfected with the I-SceI expression vector and PSVbGal, grown 4 d, and analyzed for GFP recombination and expression. (A) Genomic DNA from the two cell lines was PCR-amplified with nonrecombinant (5′-unrec) and recombinant (5′-rec) primers. The specificity of the products and the linearity of the reactions were controlled as described in Materials and Methods. qPCR of the same samples was carried out as described in Materials and Methods. (B) FACS analysis of cells transfected with I-SceI is shown. The gating of GFP + cells was created to exclude up the 99.5% of wild-type untransfected ES cells. The same gating applied to Dnmt1−/− cells shows a significant increase in the population expressing GFP. Following I-SceI transfection, Dnmt1−/− cells were treated with 5-AzadC as described in Materials and Methods. Treatment with 5-AzadC increased the fraction of cells expressing GFP in wild-type ES but did not enhance the expression of GFP in the Dnmt1−/− cells (C) The histogram showing the fraction of GFP + cells derived from three experiments is shown. To obtain reliable values of differential GFP fluorescence in ES and Dnmt1−/− cells, we compared the percentage of GFP + cells, normalized for the transfection efficiency in six experiments (three in duplicate), with the Wilcoxon Kruskal-Wallis Test, *, p
    Figure Legend Snippet: Dnmt1 Inhibits the Expression of Recombinant GFP Genes Wild-type or Dnmt1−/− ES cells carrying DR-GFP were transfected with the I-SceI expression vector and PSVbGal, grown 4 d, and analyzed for GFP recombination and expression. (A) Genomic DNA from the two cell lines was PCR-amplified with nonrecombinant (5′-unrec) and recombinant (5′-rec) primers. The specificity of the products and the linearity of the reactions were controlled as described in Materials and Methods. qPCR of the same samples was carried out as described in Materials and Methods. (B) FACS analysis of cells transfected with I-SceI is shown. The gating of GFP + cells was created to exclude up the 99.5% of wild-type untransfected ES cells. The same gating applied to Dnmt1−/− cells shows a significant increase in the population expressing GFP. Following I-SceI transfection, Dnmt1−/− cells were treated with 5-AzadC as described in Materials and Methods. Treatment with 5-AzadC increased the fraction of cells expressing GFP in wild-type ES but did not enhance the expression of GFP in the Dnmt1−/− cells (C) The histogram showing the fraction of GFP + cells derived from three experiments is shown. To obtain reliable values of differential GFP fluorescence in ES and Dnmt1−/− cells, we compared the percentage of GFP + cells, normalized for the transfection efficiency in six experiments (three in duplicate), with the Wilcoxon Kruskal-Wallis Test, *, p

    Techniques Used: Expressing, Recombinant, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, FACS, Derivative Assay, Fluorescence

    Dnmt1 Selectively Binds Recombinant GFP Chromatin Hela cells carrying DR-GFP were transfected with I-SceI and treated 24 h later with 1 μM 5-AzadC for 1, 2, and 4 d. Chromatin immunoprecipitation (ChIp) was carried out as described in Materials and Methods. (A) PCR of immunoprecipitated DNA with antibodies to Dnmt1 is shown. None indicates chromatin derived from cells transfected with control plasmid, (−) or (+) indicates the treatment with 5-AzadC. Rec, unrec indicate the primers used for amplification. The lower panel shows the statistical analysis of PCR reactions carried out at 25 and 30 cycles when the reactions with the three sets of primers were in the linear range. Immunoprecipitations were carried out with nonspecific immunoglobulin G (Control immunoglobulin G) and anti-Dnmt1 specific antibodies. The primers used were: (1) unrec; (2) rec; and (3) actin (*, p
    Figure Legend Snippet: Dnmt1 Selectively Binds Recombinant GFP Chromatin Hela cells carrying DR-GFP were transfected with I-SceI and treated 24 h later with 1 μM 5-AzadC for 1, 2, and 4 d. Chromatin immunoprecipitation (ChIp) was carried out as described in Materials and Methods. (A) PCR of immunoprecipitated DNA with antibodies to Dnmt1 is shown. None indicates chromatin derived from cells transfected with control plasmid, (−) or (+) indicates the treatment with 5-AzadC. Rec, unrec indicate the primers used for amplification. The lower panel shows the statistical analysis of PCR reactions carried out at 25 and 30 cycles when the reactions with the three sets of primers were in the linear range. Immunoprecipitations were carried out with nonspecific immunoglobulin G (Control immunoglobulin G) and anti-Dnmt1 specific antibodies. The primers used were: (1) unrec; (2) rec; and (3) actin (*, p

    Techniques Used: Recombinant, Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Derivative Assay, Plasmid Preparation, Amplification

    DNA Methylation and Recombination (A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I - SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters. Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%. (B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP + cells were analyzed by FACS. The ordinate shows the fraction of GFP + cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP + cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank ( p
    Figure Legend Snippet: DNA Methylation and Recombination (A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I - SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters. Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%. (B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP + cells were analyzed by FACS. The ordinate shows the fraction of GFP + cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP + cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank ( p

    Techniques Used: DNA Methylation Assay, Recombination Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Recombinant, Sequencing, Plasmid Preparation, Transfection, Expressing, FACS, Inhibition, Methylation, Fluorescence

    9) Product Images from "Whole-Genome DNA Array Analysis of the Response of Borrelia burgdorferi to a Bactericidal Monoclonal Antibody "

    Article Title: Whole-Genome DNA Array Analysis of the Response of Borrelia burgdorferi to a Bactericidal Monoclonal Antibody

    Journal: Infection and Immunity

    doi: 10.1128/IAI.72.4.2035-2044.2004

    Concordance of differential gene expression between DNA array and quantitative real time-PCR Analysis. Fold change values for negative controls and differentially expressed genes were generated by comparing untreated to CB2-treated samples. In all cases,
    Figure Legend Snippet: Concordance of differential gene expression between DNA array and quantitative real time-PCR Analysis. Fold change values for negative controls and differentially expressed genes were generated by comparing untreated to CB2-treated samples. In all cases,

    Techniques Used: Expressing, DNA Array, Real-time Polymerase Chain Reaction, Generated

    10) Product Images from "Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents"

    Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-018-0594-9

    Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)
    Figure Legend Snippet: Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

    Techniques Used: Methylation, Mass Spectrometry, Polymerase Chain Reaction, Derivative Assay, Positive Control, Molecular Weight

    11) Product Images from "T Cell Receptor Gene Rearrangement Lineage Analysis Reveals Clues for the Origin of Highly Restricted Antigen-specific Repertoires"

    Article Title: T Cell Receptor Gene Rearrangement Lineage Analysis Reveals Clues for the Origin of Highly Restricted Antigen-specific Repertoires

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20021945

    Single cell RT-PCR analysis reveals clusters of CW3-specific clones expressing β chains encoded by identical VDJ-β nucleotide sequences paired with different TCR-α chains. The cDNA from tubes containing single CW3-specific CD8 T cells sorted from PBL of M-2 and M-3 or M-33 was subjected to RT-PCR, and PCR products were sequenced and assigned a TCR sequence code (NS). The complete analysis is shown in Figs. S1, S2, and S3, available at http://www.jem.org/cgi/content/full/jem.20021945/DC1 . Represented here are those cells for which both a CW3-like Vβ10 sequence and a CW3-like Vα3, Vα4, or Vα8 sequence were amplified. The deduced aa sequences of the TCR junctions are shown. All TCR-α sequences incorporate the Jα35 sequence. Also shown are the number (#) of cells found for each αβ TCR clone and its corresponding percentage (%) within the αβ TCR repertoire defined here for each mouse. Each cluster of αβ TCR clones sharing an identical Vβ10 TCR nucleotide sequence is framed. Cells for which an in-frame (IF) or out of frame (OF) second TCR-α rearrangement was also amplified are indicated.
    Figure Legend Snippet: Single cell RT-PCR analysis reveals clusters of CW3-specific clones expressing β chains encoded by identical VDJ-β nucleotide sequences paired with different TCR-α chains. The cDNA from tubes containing single CW3-specific CD8 T cells sorted from PBL of M-2 and M-3 or M-33 was subjected to RT-PCR, and PCR products were sequenced and assigned a TCR sequence code (NS). The complete analysis is shown in Figs. S1, S2, and S3, available at http://www.jem.org/cgi/content/full/jem.20021945/DC1 . Represented here are those cells for which both a CW3-like Vβ10 sequence and a CW3-like Vα3, Vα4, or Vα8 sequence were amplified. The deduced aa sequences of the TCR junctions are shown. All TCR-α sequences incorporate the Jα35 sequence. Also shown are the number (#) of cells found for each αβ TCR clone and its corresponding percentage (%) within the αβ TCR repertoire defined here for each mouse. Each cluster of αβ TCR clones sharing an identical Vβ10 TCR nucleotide sequence is framed. Cells for which an in-frame (IF) or out of frame (OF) second TCR-α rearrangement was also amplified are indicated.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Expressing, Polymerase Chain Reaction, Sequencing, Amplification

    Repertoire analysis by single cell DNA-PCR for coamplification of CW3-specific TCR VDJ-β and VJ-α sequences together with DJ-β rearrangements as potential clonal markers. For each of three mice (M-41, M-42, and M-43), single pCW3Kd + Vβ10 + CD8 + splenocytes were sorted 2 wk after immunization with P815-CW3 tumor cells. The rearranged TCR-α and TCR-β nucleotide sequences were amplified by single cell DNA-PCR. PCR products from cells with successful amplifications for TCR-α and TCR-β rearrangements were sequenced to identify paired αβ TCRs. Other details are as described for Fig. 2 .
    Figure Legend Snippet: Repertoire analysis by single cell DNA-PCR for coamplification of CW3-specific TCR VDJ-β and VJ-α sequences together with DJ-β rearrangements as potential clonal markers. For each of three mice (M-41, M-42, and M-43), single pCW3Kd + Vβ10 + CD8 + splenocytes were sorted 2 wk after immunization with P815-CW3 tumor cells. The rearranged TCR-α and TCR-β nucleotide sequences were amplified by single cell DNA-PCR. PCR products from cells with successful amplifications for TCR-α and TCR-β rearrangements were sequenced to identify paired αβ TCRs. Other details are as described for Fig. 2 .

    Techniques Used: Polymerase Chain Reaction, Mouse Assay, Amplification

    12) Product Images from "A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression"

    Article Title: A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063618

    Colony PCR showing the cDNA derived maternal and paternal fragment of Nvtra . Panels A – D and E – H indicate the four replicate PCR runs. The maternal origin and age of the embryos is indicated above each set of PCR fragments. White arrow indicates cDNA fragment, black arrow indicates Russia Bait cDNA fragment and dotted arrow indicates gDNA fragment. M is 100 bp molecular standard, ranging from 300 bp (lowest band) to 500 bp (higest band).
    Figure Legend Snippet: Colony PCR showing the cDNA derived maternal and paternal fragment of Nvtra . Panels A – D and E – H indicate the four replicate PCR runs. The maternal origin and age of the embryos is indicated above each set of PCR fragments. White arrow indicates cDNA fragment, black arrow indicates Russia Bait cDNA fragment and dotted arrow indicates gDNA fragment. M is 100 bp molecular standard, ranging from 300 bp (lowest band) to 500 bp (higest band).

    Techniques Used: Polymerase Chain Reaction, Derivative Assay

    13) Product Images from "Acquired resistance to PI3K/mTOR inhibition is associated with mitochondrial DNA mutation and glycolysis"

    Article Title: Acquired resistance to PI3K/mTOR inhibition is associated with mitochondrial DNA mutation and glycolysis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22655

    ( A ) Representation of somatic mitochondrial DNA variants detected only in AQR clones in MT-CO1 gene. ( B ) Validation of the ENST00000361624.2: c.1367T > A variant by pyrosequencing. ( C ) PCR verification of ρ(o) status of H1975ρ(o) cells compared with parental H1975 cells. ( D ) Bar charts showing IC50 values of BEZ235, PI103, KU-0063794 and carboplatin in H1975 (blue columns) and H1975ρ0 (black columns) cells. Also shown are the levels of extracellular lactate and reactive oxygen species in H1975 and H1975ρ0 cells. * p
    Figure Legend Snippet: ( A ) Representation of somatic mitochondrial DNA variants detected only in AQR clones in MT-CO1 gene. ( B ) Validation of the ENST00000361624.2: c.1367T > A variant by pyrosequencing. ( C ) PCR verification of ρ(o) status of H1975ρ(o) cells compared with parental H1975 cells. ( D ) Bar charts showing IC50 values of BEZ235, PI103, KU-0063794 and carboplatin in H1975 (blue columns) and H1975ρ0 (black columns) cells. Also shown are the levels of extracellular lactate and reactive oxygen species in H1975 and H1975ρ0 cells. * p

    Techniques Used: Clone Assay, Variant Assay, Polymerase Chain Reaction

    14) Product Images from "Making (anti-) sense out of huntingtin levels in Huntington disease"

    Article Title: Making (anti-) sense out of huntingtin levels in Huntington disease

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-015-0018-7

    Validating RT-PCR amplification across the CAG repeat in HD patient-derived fibroblasts. Wild-type and mutant HTT were separated by gel electrophoresis. (A) Standard curve of wild-type and mutant HTT RT-PCR products with increasing PCR cycles from gDNA derived from post-mortem brain tissue of 7 HD patients. PCR linearity was evaluated by determining the individual linear regression coefficients ( r 2 ) of the band intensities of wild-type and mutant HTT expression versus the number of PCR cycles, n = 7. (B) PCR products from cDNA of 4 HD (GM00305, GM02173, GM04022, GM04855) fibroblasts. CAG repeat sizes for the wild-type (lower band) and mutant alleles (upper band) are indicated below each lane. gDNA was used to examine differences in PCR amplification between the wild-type and mutant product due to the CAG repeat expansion. (C) RT-PCR products with input: cDNA (+RT), cDNA lacking reverse transcriptase (−RT) and gDNA of one control (GM04204). (D) Whisker boxplot of RT-PCR from HD patient-derived fibroblasts, comparing wild-type and mutant HTT mRNA expression levels, relative to gDNA. Line = mean, pair wise differences were evaluated using linear mixed model, n = 4.
    Figure Legend Snippet: Validating RT-PCR amplification across the CAG repeat in HD patient-derived fibroblasts. Wild-type and mutant HTT were separated by gel electrophoresis. (A) Standard curve of wild-type and mutant HTT RT-PCR products with increasing PCR cycles from gDNA derived from post-mortem brain tissue of 7 HD patients. PCR linearity was evaluated by determining the individual linear regression coefficients ( r 2 ) of the band intensities of wild-type and mutant HTT expression versus the number of PCR cycles, n = 7. (B) PCR products from cDNA of 4 HD (GM00305, GM02173, GM04022, GM04855) fibroblasts. CAG repeat sizes for the wild-type (lower band) and mutant alleles (upper band) are indicated below each lane. gDNA was used to examine differences in PCR amplification between the wild-type and mutant product due to the CAG repeat expansion. (C) RT-PCR products with input: cDNA (+RT), cDNA lacking reverse transcriptase (−RT) and gDNA of one control (GM04204). (D) Whisker boxplot of RT-PCR from HD patient-derived fibroblasts, comparing wild-type and mutant HTT mRNA expression levels, relative to gDNA. Line = mean, pair wise differences were evaluated using linear mixed model, n = 4.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Derivative Assay, Mutagenesis, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Expressing, Whisker Assay

    15) Product Images from "Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation"

    Article Title: Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation

    Journal: Oncogene

    doi: 10.1038/onc.2011.319

    Methylation status of the rRNA promoter in the parental colorectal carcinoma cells HCT116 and its derivative knockout cell lines. The methylation status of each CpG dinucleotide spanning −158 to +29 bp of human rRNA promoter in HCT116 colorectal carcinoma cells and derivative lines with targeted disruption of DNMT1 , DNMT3b or both genes. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 16–19 randomly selected clones from each sample were subjected to automated sequencing. Each row represents the sequence of an individual clone, whereas each column depicts the position of the CpG. The filled and open circles denote methylated and unmethylated CpGs, respectively. The top row shows a heat map of methylated CpGs representing the frequency of methylation in each of the individual clones shown below. The degree of hypermethylation (NIM) at three CpG islands was assessed using quantitative real-time methylation-specific PCR and was normalized to the amount of bisulfite-converted input as described in ‘Materials and methods.'
    Figure Legend Snippet: Methylation status of the rRNA promoter in the parental colorectal carcinoma cells HCT116 and its derivative knockout cell lines. The methylation status of each CpG dinucleotide spanning −158 to +29 bp of human rRNA promoter in HCT116 colorectal carcinoma cells and derivative lines with targeted disruption of DNMT1 , DNMT3b or both genes. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 16–19 randomly selected clones from each sample were subjected to automated sequencing. Each row represents the sequence of an individual clone, whereas each column depicts the position of the CpG. The filled and open circles denote methylated and unmethylated CpGs, respectively. The top row shows a heat map of methylated CpGs representing the frequency of methylation in each of the individual clones shown below. The degree of hypermethylation (NIM) at three CpG islands was assessed using quantitative real-time methylation-specific PCR and was normalized to the amount of bisulfite-converted input as described in ‘Materials and methods.'

    Techniques Used: Methylation, Knock-Out, Amplification, Clone Assay, Sequencing, Polymerase Chain Reaction

    16) Product Images from "Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma"

    Article Title: Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

    Journal: International Journal of Otolaryngology

    doi: 10.1155/2009/848695

    Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.
    Figure Legend Snippet: Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

    Techniques Used: Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Positive Control, Marker, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis

    17) Product Images from "Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups ▿"

    Article Title: Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups ▿

    Journal:

    doi: 10.1128/AEM.00370-09

    PCR-RFLP analysis of the rRNA IGS region for differentiating the lineages of F. oxysporum f. sp. cubense . For this procedure, DNA obtained from a putative F. oxysporum f. sp. cubense isolate is used as a template for amplication of the IGS region (A),
    Figure Legend Snippet: PCR-RFLP analysis of the rRNA IGS region for differentiating the lineages of F. oxysporum f. sp. cubense . For this procedure, DNA obtained from a putative F. oxysporum f. sp. cubense isolate is used as a template for amplication of the IGS region (A),

    Techniques Used: Polymerase Chain Reaction

    18) Product Images from "Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor"

    Article Title: Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor

    Journal: Nuclear Receptor

    doi: 10.1186/1478-1336-2-1

    CAR expression and alternative splicing of exon 9 during enterocytic differentiation of Caco-2 TC7 cells . (A) Northern Blot analysis with polyadenylated RNA of the indicated cell lines. Caco-2 TC7 cells were analyzed from subconfluent (sub), confluent (confl) and 15 days post-confluent (15d pc) cultures. The blot was sequentially hybridized with probes for the genes indicated. The arrow marks the major CAR mRNA species of 1.4 to 1.7 kb. (B) Analysis of the expression of transcripts with alternatively spliced exon 9 by qualitative RT-PCR with random hexamer primed cDNA of polyadenylated RNA of Caco-2 TC7 cells cultured until confluence (confl) and for 15 days post-confluent (15d pc). PCR was performed with primers F2/R2 (Table 1 ). SV1 and SV5 denote control reactions performed with DNA of the corresponding CAR isoform expression plasmids. The lane on the left shows a 50 bp ladder size marker. By mixing DNA of SV1 and SV5 CAR isoform expression plasmids in different molar ratios, we confirmed that both fragments were amplified with equal efficiency (data not shown).
    Figure Legend Snippet: CAR expression and alternative splicing of exon 9 during enterocytic differentiation of Caco-2 TC7 cells . (A) Northern Blot analysis with polyadenylated RNA of the indicated cell lines. Caco-2 TC7 cells were analyzed from subconfluent (sub), confluent (confl) and 15 days post-confluent (15d pc) cultures. The blot was sequentially hybridized with probes for the genes indicated. The arrow marks the major CAR mRNA species of 1.4 to 1.7 kb. (B) Analysis of the expression of transcripts with alternatively spliced exon 9 by qualitative RT-PCR with random hexamer primed cDNA of polyadenylated RNA of Caco-2 TC7 cells cultured until confluence (confl) and for 15 days post-confluent (15d pc). PCR was performed with primers F2/R2 (Table 1 ). SV1 and SV5 denote control reactions performed with DNA of the corresponding CAR isoform expression plasmids. The lane on the left shows a 50 bp ladder size marker. By mixing DNA of SV1 and SV5 CAR isoform expression plasmids in different molar ratios, we confirmed that both fragments were amplified with equal efficiency (data not shown).

    Techniques Used: Expressing, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Random Hexamer Labeling, Cell Culture, Polymerase Chain Reaction, Marker, Amplification

    Tissue-specific splicing of exons 8 and 9 . Qualitative RT-PCR analysis of the expression of transcripts with alternatively spliced exon 8 (A) and exon 9 (B) in the indicated human tissues. Small intestine and colon mucosa samples were derived from one individual each. In contrast, the samples of the other tissues represent pools (Human Total RNA Master Panel II, Clontech). The assays were performed as described in Experimental Procedures, with random hexamer primed cDNA of total RNA and primer pairs F1/R1 (A) or F2/R2 (B) (see Table 1 ). SV1, SV2, SV5 denote control reactions performed with DNA of the corresponding CAR isoform expression plasmids.
    Figure Legend Snippet: Tissue-specific splicing of exons 8 and 9 . Qualitative RT-PCR analysis of the expression of transcripts with alternatively spliced exon 8 (A) and exon 9 (B) in the indicated human tissues. Small intestine and colon mucosa samples were derived from one individual each. In contrast, the samples of the other tissues represent pools (Human Total RNA Master Panel II, Clontech). The assays were performed as described in Experimental Procedures, with random hexamer primed cDNA of total RNA and primer pairs F1/R1 (A) or F2/R2 (B) (see Table 1 ). SV1, SV2, SV5 denote control reactions performed with DNA of the corresponding CAR isoform expression plasmids.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Random Hexamer Labeling

    19) Product Images from "Importance of the Long-Chain Fatty Acid Beta-Hydroxylating Cytochrome P450 Enzyme YbdT for Lipopeptide Biosynthesis in Bacillus subtilis Strain OKB105"

    Article Title: Importance of the Long-Chain Fatty Acid Beta-Hydroxylating Cytochrome P450 Enzyme YbdT for Lipopeptide Biosynthesis in Bacillus subtilis Strain OKB105

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms12031767

    The overlap PCR protocol used to generate a ybdT mutation in OKB105. Primers P1 and P2 amplified the 5′ region of ybdT gene in OKB105. Primers P5 and P6 amplified the 3′ region of ybdT gene. Primers P3 and P4 amplified the chloramphenicol resistance cassette from PDG1662. Primers P2 P3, P4 and P5 were designed with 18 bp overlap as shown in Table 4 . PCR1, PCR2, and PCR3 are the primary PCR products with overlapping region as follows: the 3′end of PCR1 contains sequence for the upstream portion of the cat and the 5′ end of PCR3 has sequence for amino acids 149–151 encoded by ybdT (blue). The 5′end of PCR2 contains sequence for amino acids 214–226 encoded by cat and 3′end of PCR3 has sequence for amino acids 365–367 encoded by ybdT (red). The primary PCR products were then joined in a long PCR reaction to synthesize the PCR construct used for transforming competent OKB105 cells.
    Figure Legend Snippet: The overlap PCR protocol used to generate a ybdT mutation in OKB105. Primers P1 and P2 amplified the 5′ region of ybdT gene in OKB105. Primers P5 and P6 amplified the 3′ region of ybdT gene. Primers P3 and P4 amplified the chloramphenicol resistance cassette from PDG1662. Primers P2 P3, P4 and P5 were designed with 18 bp overlap as shown in Table 4 . PCR1, PCR2, and PCR3 are the primary PCR products with overlapping region as follows: the 3′end of PCR1 contains sequence for the upstream portion of the cat and the 5′ end of PCR3 has sequence for amino acids 149–151 encoded by ybdT (blue). The 5′end of PCR2 contains sequence for amino acids 214–226 encoded by cat and 3′end of PCR3 has sequence for amino acids 365–367 encoded by ybdT (red). The primary PCR products were then joined in a long PCR reaction to synthesize the PCR construct used for transforming competent OKB105 cells.

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Amplification, Sequencing, Construct

    20) Product Images from "Tissue Microdissection and Degenerate Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) Is an Effective Method to Analyze Genetic Aberrations in Invasive Tumors"

    Article Title: Tissue Microdissection and Degenerate Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) Is an Effective Method to Analyze Genetic Aberrations in Invasive Tumors

    Journal: The Journal of molecular diagnostics : JMD

    doi:

    Ratio profiles from DNA prepared from unfixed SF210 human glioblastoma cells. The x -axis represents the position along the chromosome (p arm to the left and q arm to the right). The centromeres are marked by a crosshatch on the x -axis. The y -axis represents normalized test/reference fluorescence intensity ratios (copy number aberrations). The mean and SD of the fluorescence intensity ratios for the indicated chromosomes are shown. All chromosomal aberrations in experiments using DNA extracted without amplification and labeled by nick translation ( A ) were seen in those with DOP-PCR product derived from 50 pg ( B ) to 250 ng DNA (data not shown). However, experiments performed with DOP-PCR product from 10 pg DNA ( C ) did not show gain on chromosomal arm 6p ( arrowheads ), and ratio profiles were of poor quality (note the large standard deviations). There were no aberrations on chromosomes not shown in the figure.
    Figure Legend Snippet: Ratio profiles from DNA prepared from unfixed SF210 human glioblastoma cells. The x -axis represents the position along the chromosome (p arm to the left and q arm to the right). The centromeres are marked by a crosshatch on the x -axis. The y -axis represents normalized test/reference fluorescence intensity ratios (copy number aberrations). The mean and SD of the fluorescence intensity ratios for the indicated chromosomes are shown. All chromosomal aberrations in experiments using DNA extracted without amplification and labeled by nick translation ( A ) were seen in those with DOP-PCR product derived from 50 pg ( B ) to 250 ng DNA (data not shown). However, experiments performed with DOP-PCR product from 10 pg DNA ( C ) did not show gain on chromosomal arm 6p ( arrowheads ), and ratio profiles were of poor quality (note the large standard deviations). There were no aberrations on chromosomes not shown in the figure.

    Techniques Used: Fluorescence, Amplification, Labeling, Nick Translation, Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Derivative Assay

    Agarose gel electrophoresis of DOP-PCR products. Five microliters of each DOP-PCR product were applied to 1.2% agarose gel. M, φX 174/ Hae III. The sizes of the molecular weight markers are indicated at the left. Lanes 1 - 6 : DOP-PCR product from 10 pg ( 1 ), 100 pg ( 2 ), 1 ng ( 3 ), 10 ng ( 4 ), 50 ng ( 5 ), and 250 ng ( 6 ) of DNA from unfixed U251NCI cells. Size of DOP-PCR products ranged from approximately 400 to 1500 bp, and amplification was better when template DNA was 1 ng or more. Lanes 7 - 11 show DOP-PCR product from DNA extracted from fixed, embedded, and microdissected piece of MG-stained ( 7 and 8 ) and HE-stained ( 9–11 ) U251NCI cell pellet. DNA s were extracted from smaller (2 mm × 2 mm, lanes 7 and 9 ) and larger (6 mm × 6 mm; lanes 8 , 10 , and 11 ) pieces in the same amount of buffer (60 μl) and amplified by DOP-PCR. DNA from the larger HE piece was amplified with ( lane 10 ) or without ( lane 11 ) increased magnesium. Size of DOP-PCR products from fixed and microdissected samples (apparently
    Figure Legend Snippet: Agarose gel electrophoresis of DOP-PCR products. Five microliters of each DOP-PCR product were applied to 1.2% agarose gel. M, φX 174/ Hae III. The sizes of the molecular weight markers are indicated at the left. Lanes 1 - 6 : DOP-PCR product from 10 pg ( 1 ), 100 pg ( 2 ), 1 ng ( 3 ), 10 ng ( 4 ), 50 ng ( 5 ), and 250 ng ( 6 ) of DNA from unfixed U251NCI cells. Size of DOP-PCR products ranged from approximately 400 to 1500 bp, and amplification was better when template DNA was 1 ng or more. Lanes 7 - 11 show DOP-PCR product from DNA extracted from fixed, embedded, and microdissected piece of MG-stained ( 7 and 8 ) and HE-stained ( 9–11 ) U251NCI cell pellet. DNA s were extracted from smaller (2 mm × 2 mm, lanes 7 and 9 ) and larger (6 mm × 6 mm; lanes 8 , 10 , and 11 ) pieces in the same amount of buffer (60 μl) and amplified by DOP-PCR. DNA from the larger HE piece was amplified with ( lane 10 ) or without ( lane 11 ) increased magnesium. Size of DOP-PCR products from fixed and microdissected samples (apparently

    Techniques Used: Agarose Gel Electrophoresis, Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Molecular Weight, Amplification, Staining

    Ratio profiles from DNA prepared from unfixed U251NCI human glioblastoma cells. All chromosomal aberrations in experiments using DNA extracted without amplification and labeled by nick translation ( A ) were seen in those with DOP-PCR product from 50 pg ( B ) to 250 ng DNA (data not shown). Experiments performed with DOP-PCR product from 10 pg DNA ( C ) did not show gains on chromosomal arm 9p ( arrowheads ), and ratio profiles were of poor quality (note the large standard deviations). There were no aberrations on the chromosomes not shown in the figure.
    Figure Legend Snippet: Ratio profiles from DNA prepared from unfixed U251NCI human glioblastoma cells. All chromosomal aberrations in experiments using DNA extracted without amplification and labeled by nick translation ( A ) were seen in those with DOP-PCR product from 50 pg ( B ) to 250 ng DNA (data not shown). Experiments performed with DOP-PCR product from 10 pg DNA ( C ) did not show gains on chromosomal arm 9p ( arrowheads ), and ratio profiles were of poor quality (note the large standard deviations). There were no aberrations on the chromosomes not shown in the figure.

    Techniques Used: Amplification, Labeling, Nick Translation, Degenerate Oligonucleotide–primed Polymerase Chain Reaction

    Ratio profiles from fixed and microdissected human mixed oligo-astrocytoma. DNAs were extracted from histologically neoplastic ( * ) and normal ( ** ) regions and amplified by DOP-PCR. A: MG-stained section where microdissected areas are indicated by the dotted squares. B: HE-stained section where areas to be microdissected are indicated by the dotted squares. C: CGH ratio profile from histologically normal and neoplastic regions. The former showed no aberrations, whereas the latter showed losses on chromosome arms 1p and 19q.
    Figure Legend Snippet: Ratio profiles from fixed and microdissected human mixed oligo-astrocytoma. DNAs were extracted from histologically neoplastic ( * ) and normal ( ** ) regions and amplified by DOP-PCR. A: MG-stained section where microdissected areas are indicated by the dotted squares. B: HE-stained section where areas to be microdissected are indicated by the dotted squares. C: CGH ratio profile from histologically normal and neoplastic regions. The former showed no aberrations, whereas the latter showed losses on chromosome arms 1p and 19q.

    Techniques Used: Amplification, Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Staining

    21) Product Images from "Expression of a Heterologous S-Adenosylmethionine Decarboxylase cDNA in Plants Demonstrates That Changes in S-Adenosyl-l-Methionine Decarboxylase Activity Determine Levels of the Higher Polyamines Spermidine and Spermine 1"

    Article Title: Expression of a Heterologous S-Adenosylmethionine Decarboxylase cDNA in Plants Demonstrates That Changes in S-Adenosyl-l-Methionine Decarboxylase Activity Determine Levels of the Higher Polyamines Spermidine and Spermine 1

    Journal: Plant Physiology

    doi: 10.1104/pp.010966

    Generation and molecular characterization of transgenic rice plants expressing the D. stramonium samdc cDNA. A, Map of Ubi::Dsamdc showing transcription unit, relevant restriction sites, and primers used for PCR and RT-PCR analyses. The D. stramonium samdc cDNA is 1.839 kb in size. Kpn I has a single restriction site in the plasmid. Nos, Nopaline synthase. Arrows represent primers and length of amplified fragment. B, DNA gel-blot analysis of transgenic rice plants. Genomic DNA (10 μg) was digested with Kpn I and probed with the 0.9-kb DIG-labeled PCR product from Ubi::Dsamdc . Exposure time was 10 min; wt, wild type; numbers represent putative transgenic plants; L, molecular size marker (1-kb DNA ladder, Invitrogen, Carlsbad, CA). C, RT-PCR analysis of D. stramonium samdc cDNA (0.9 kb) from total RNA extracted from controls and plants transformed with Ubi::Dsamdc . L, Molecular size marker (1-kb DNA ladder, Invitrogen); +ve, positive control, plasmid Ubi::Dsamdc ; −ve, negative control (water); numbers indicate independent transgenic plants; wt, wild type. D, RT-PCR analysis of rice samdc from total RNA extracted from controls and plants transformed with Ubi::Dsamdc. L, Molecular size marker (1-kb DNA ladder, Invitrogen); +ve, positive control, plasmid Ubi::Dsamdc ; −ve, negative control (water); numbers represent indicate independent transgenic plants; wt, wild type.
    Figure Legend Snippet: Generation and molecular characterization of transgenic rice plants expressing the D. stramonium samdc cDNA. A, Map of Ubi::Dsamdc showing transcription unit, relevant restriction sites, and primers used for PCR and RT-PCR analyses. The D. stramonium samdc cDNA is 1.839 kb in size. Kpn I has a single restriction site in the plasmid. Nos, Nopaline synthase. Arrows represent primers and length of amplified fragment. B, DNA gel-blot analysis of transgenic rice plants. Genomic DNA (10 μg) was digested with Kpn I and probed with the 0.9-kb DIG-labeled PCR product from Ubi::Dsamdc . Exposure time was 10 min; wt, wild type; numbers represent putative transgenic plants; L, molecular size marker (1-kb DNA ladder, Invitrogen, Carlsbad, CA). C, RT-PCR analysis of D. stramonium samdc cDNA (0.9 kb) from total RNA extracted from controls and plants transformed with Ubi::Dsamdc . L, Molecular size marker (1-kb DNA ladder, Invitrogen); +ve, positive control, plasmid Ubi::Dsamdc ; −ve, negative control (water); numbers indicate independent transgenic plants; wt, wild type. D, RT-PCR analysis of rice samdc from total RNA extracted from controls and plants transformed with Ubi::Dsamdc. L, Molecular size marker (1-kb DNA ladder, Invitrogen); +ve, positive control, plasmid Ubi::Dsamdc ; −ve, negative control (water); numbers represent indicate independent transgenic plants; wt, wild type.

    Techniques Used: Transgenic Assay, Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Amplification, Western Blot, Labeling, Marker, Transformation Assay, Positive Control, Negative Control

    22) Product Images from "Acquired resistance to PI3K/mTOR inhibition is associated with mitochondrial DNA mutation and glycolysis"

    Article Title: Acquired resistance to PI3K/mTOR inhibition is associated with mitochondrial DNA mutation and glycolysis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22655

    ( A ) Representation of somatic mitochondrial DNA variants detected only in AQR clones in MT-CO1 gene. ( B ) Validation of the ENST00000361624.2: c.1367T > A variant by pyrosequencing. ( C ) PCR verification of ρ(o) status of H1975ρ(o) cells compared with parental H1975 cells. ( D ) Bar charts showing IC50 values of BEZ235, PI103, KU-0063794 and carboplatin in H1975 (blue columns) and H1975ρ0 (black columns) cells. Also shown are the levels of extracellular lactate and reactive oxygen species in H1975 and H1975ρ0 cells. * p
    Figure Legend Snippet: ( A ) Representation of somatic mitochondrial DNA variants detected only in AQR clones in MT-CO1 gene. ( B ) Validation of the ENST00000361624.2: c.1367T > A variant by pyrosequencing. ( C ) PCR verification of ρ(o) status of H1975ρ(o) cells compared with parental H1975 cells. ( D ) Bar charts showing IC50 values of BEZ235, PI103, KU-0063794 and carboplatin in H1975 (blue columns) and H1975ρ0 (black columns) cells. Also shown are the levels of extracellular lactate and reactive oxygen species in H1975 and H1975ρ0 cells. * p

    Techniques Used: Clone Assay, Variant Assay, Polymerase Chain Reaction

    23) Product Images from "Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site"

    Article Title: Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site

    Journal: Nucleic Acids Research

    doi:

    MDR1 quantification using SRS-cPCR. Competitive PCR and restriction digest were carried out as described in Materials and Methods. Lanes 1 and 12 in each panel contain size marker (100 bp ladder). Lanes 10 and 11 represent amplification products of only competitor and target mRNA, respectively. ( A ) Twenty microliters PCR product was loaded on a 2% agarose gel. The target and the competitor products were identical in size (299 bp). Lanes 2–9 contain 10 6 molecules of an in vitro transcribed cloned human wild-type MDR1 fragment and a semi-logarithmic dilution of the competitor RNA ranging from 10 4 to 3 × 10 7 molecules . ( B ) After digestion with the restriction endonuclease Pvu II, the wild-type PCR products were cleaved into 127 and 172 bp fragments, and the competitor PCR products into 100 and 199 bp fragments. The EQP is indicated by an arrow (lane 5). It was determined by comparison of signal intensities of the 100 and 127 bp fragments, or of the 172 and 199 bp fragments, respectively. The competitive amplification of an external control gene, β 2-MG , permitting correction of target quantification (29) is not displayed. In competitive PCR reactions, the subdominant template yields visible products if it represents at least 1% of the total. Three competitor dilutions covering a range of 3 logs (lanes 3, 6 and 9, indicated by arrows) usually permit the visualization of target- and competitor-derived products in at least one of the reactions (lanes 3 and 6), thus permitting calculation of the target molecules. ( C ) The band intensities of competitor- and target-derived fragments were digitalized and measured by using the 1D Image Analysis Software. In the graph, the mean intensities from the larger competitor (line B) and the target (line A) fragments were used for the performance of a regression analysis. The EQP is indicated by the intersection of the two lines. The numbers on the abscissa correspond to the lane numbers on the agarose gel. Elimination of any data points between the extreme values has no effect on the EQP. This demonstrates that it is possible to reduce the number of competitor dilutions needed for quantitative analysis of target transcripts, thus rendering the assay less laborious.
    Figure Legend Snippet: MDR1 quantification using SRS-cPCR. Competitive PCR and restriction digest were carried out as described in Materials and Methods. Lanes 1 and 12 in each panel contain size marker (100 bp ladder). Lanes 10 and 11 represent amplification products of only competitor and target mRNA, respectively. ( A ) Twenty microliters PCR product was loaded on a 2% agarose gel. The target and the competitor products were identical in size (299 bp). Lanes 2–9 contain 10 6 molecules of an in vitro transcribed cloned human wild-type MDR1 fragment and a semi-logarithmic dilution of the competitor RNA ranging from 10 4 to 3 × 10 7 molecules . ( B ) After digestion with the restriction endonuclease Pvu II, the wild-type PCR products were cleaved into 127 and 172 bp fragments, and the competitor PCR products into 100 and 199 bp fragments. The EQP is indicated by an arrow (lane 5). It was determined by comparison of signal intensities of the 100 and 127 bp fragments, or of the 172 and 199 bp fragments, respectively. The competitive amplification of an external control gene, β 2-MG , permitting correction of target quantification (29) is not displayed. In competitive PCR reactions, the subdominant template yields visible products if it represents at least 1% of the total. Three competitor dilutions covering a range of 3 logs (lanes 3, 6 and 9, indicated by arrows) usually permit the visualization of target- and competitor-derived products in at least one of the reactions (lanes 3 and 6), thus permitting calculation of the target molecules. ( C ) The band intensities of competitor- and target-derived fragments were digitalized and measured by using the 1D Image Analysis Software. In the graph, the mean intensities from the larger competitor (line B) and the target (line A) fragments were used for the performance of a regression analysis. The EQP is indicated by the intersection of the two lines. The numbers on the abscissa correspond to the lane numbers on the agarose gel. Elimination of any data points between the extreme values has no effect on the EQP. This demonstrates that it is possible to reduce the number of competitor dilutions needed for quantitative analysis of target transcripts, thus rendering the assay less laborious.

    Techniques Used: Polymerase Chain Reaction, Marker, Amplification, Agarose Gel Electrophoresis, In Vitro, Clone Assay, Derivative Assay, Software

    24) Product Images from "Small Variant STEVOR Antigen Is Uniquely Located within Maurer's Clefts in Plasmodium falciparum-Infected Red Blood Cells"

    Article Title: Small Variant STEVOR Antigen Is Uniquely Located within Maurer's Clefts in Plasmodium falciparum-Infected Red Blood Cells

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.1.6.926-935.2002

    (A) Schematic representation of stevor (1,000 bp). The external (smkf1 and smkr1) and internal (RepF1, RepF2, and RepR) primers used in this study are indicated. Dark grey shading, moderately polymorphic. Light grey shading, highly polymorphic. Broken line, weakly predicted transmembrane domain. Solid line (above sequence and at right), strongly predicted transmembrane domain. AS, position of alternative splice site. Positions of peptides (Pep) 1 and 2 are also shown. (B) PCR and RT-PCR analyses of stevor . (I) PCR products of 3D7 genomic DNA amplified with smkf1 and smkr1 and with RepF1, RepF2, and RepR. (II) RT-PCR products obtained from RNA of asynchronous parasites with smkf1 and smkr1 and with RepF1, RepF2, and RepR. The products were separated on Metaphor 3% agarose gels. Lanes +, RT added. Lanes −, no RT added. Lanes M, 100-bp ladder (Bio-Rad). (C) Southern blot analysis of P. falciparum 3D7 chromosomes probed with PCR or RT-PCR products. The left panel shows PFGE of 3D7 chromosomes stained with ethidium bromide (EtBr) and photographed before the DNA from the gels was transferred to nylon membranes and probed. Numbers at left refer to P. falciparum chromosomes. Probing was performed with the PCR product obtained from DNA (middle panel) and with the RT-PCR product obtained from RNA (cDNA) (right panel) of 3D7 parasites.
    Figure Legend Snippet: (A) Schematic representation of stevor (1,000 bp). The external (smkf1 and smkr1) and internal (RepF1, RepF2, and RepR) primers used in this study are indicated. Dark grey shading, moderately polymorphic. Light grey shading, highly polymorphic. Broken line, weakly predicted transmembrane domain. Solid line (above sequence and at right), strongly predicted transmembrane domain. AS, position of alternative splice site. Positions of peptides (Pep) 1 and 2 are also shown. (B) PCR and RT-PCR analyses of stevor . (I) PCR products of 3D7 genomic DNA amplified with smkf1 and smkr1 and with RepF1, RepF2, and RepR. (II) RT-PCR products obtained from RNA of asynchronous parasites with smkf1 and smkr1 and with RepF1, RepF2, and RepR. The products were separated on Metaphor 3% agarose gels. Lanes +, RT added. Lanes −, no RT added. Lanes M, 100-bp ladder (Bio-Rad). (C) Southern blot analysis of P. falciparum 3D7 chromosomes probed with PCR or RT-PCR products. The left panel shows PFGE of 3D7 chromosomes stained with ethidium bromide (EtBr) and photographed before the DNA from the gels was transferred to nylon membranes and probed. Numbers at left refer to P. falciparum chromosomes. Probing was performed with the PCR product obtained from DNA (middle panel) and with the RT-PCR product obtained from RNA (cDNA) (right panel) of 3D7 parasites.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Southern Blot, Staining

    25) Product Images from "Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿ †"

    Article Title: Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00040-10

    Dendrograms showing the similarity of PCR-DGGE profiles generated with the bacterial 16S rRNA DGGE system for soil B at the flowering (A) and senescence (B) stages and for soil V at the flowering stage (C). B, Buinen soil; A, cultivar Aveka; Av, cultivar
    Figure Legend Snippet: Dendrograms showing the similarity of PCR-DGGE profiles generated with the bacterial 16S rRNA DGGE system for soil B at the flowering (A) and senescence (B) stages and for soil V at the flowering stage (C). B, Buinen soil; A, cultivar Aveka; Av, cultivar

    Techniques Used: Polymerase Chain Reaction, Denaturing Gradient Gel Electrophoresis, Generated

    26) Product Images from "Protective Efficacy of VP1-Specific Neutralizing Antibody Associated with a Reduction of Viral Load and Pro-Inflammatory Cytokines in Human SCARB2-Transgenic Mice"

    Article Title: Protective Efficacy of VP1-Specific Neutralizing Antibody Associated with a Reduction of Viral Load and Pro-Inflammatory Cytokines in Human SCARB2-Transgenic Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0069858

    N3-mediated neutralization against B4 genotype of E59 and C2 genotype of 5746 viruses in vitro. ( A ) Both E59 and 5746 were pre-incubated (m.o.i. = 0.5) with various amounts of N3 for 1 h at 37°C before adding them to 3T3-SCARB2 cells. RNA was extracted 2 h after infection, and subjected to RT-PCR to detect the expression of viral genome P1. The amounts of N3 in different lanes were Lane 1∶50 µg, Lane 2∶10 µg, Lane 3∶2 µg, Lane 4∶0.4 µg, Lane 5: uninfected 3T3-SCARB2 cells as the negative control, and Lane 6: E59 viral cDNA as the positive control were included. The same loading scheme was applied to ( B ) where Lane 6 was replaced by 5746 virus for the control. Expression of cytosolic GADPH as the internal control of RT-PCR was detected. ( C ) and ( D ) The bar graph represents the densitometric quantification of the band intensities of viral genome P1 and GADPH from ( A ) and ( B ), respectively. The error bar of each group generated from three independent experiments was included.
    Figure Legend Snippet: N3-mediated neutralization against B4 genotype of E59 and C2 genotype of 5746 viruses in vitro. ( A ) Both E59 and 5746 were pre-incubated (m.o.i. = 0.5) with various amounts of N3 for 1 h at 37°C before adding them to 3T3-SCARB2 cells. RNA was extracted 2 h after infection, and subjected to RT-PCR to detect the expression of viral genome P1. The amounts of N3 in different lanes were Lane 1∶50 µg, Lane 2∶10 µg, Lane 3∶2 µg, Lane 4∶0.4 µg, Lane 5: uninfected 3T3-SCARB2 cells as the negative control, and Lane 6: E59 viral cDNA as the positive control were included. The same loading scheme was applied to ( B ) where Lane 6 was replaced by 5746 virus for the control. Expression of cytosolic GADPH as the internal control of RT-PCR was detected. ( C ) and ( D ) The bar graph represents the densitometric quantification of the band intensities of viral genome P1 and GADPH from ( A ) and ( B ), respectively. The error bar of each group generated from three independent experiments was included.

    Techniques Used: Neutralization, In Vitro, Incubation, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Positive Control, Generated

    27) Product Images from "Methylation profiling of SOCS1, SOCS2, SOCS3, CISH and SHP1 in Philadelphia-negative myeloproliferative neoplasm"

    Article Title: Methylation profiling of SOCS1, SOCS2, SOCS3, CISH and SHP1 in Philadelphia-negative myeloproliferative neoplasm

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12103

    Methylation-specific polymerase chain reaction of SOCS1 , SOCS2 -5′, SOCS3, CISH and SHP1 in cell lines. For SHP1 , K562 was homozygously methylated, while HEL, SET-2 and MEG-01 were completely unmethylated. HEL, SET-2, MEG-01 and K562 were completely unmethylated for SOCS1 , SOCS2 -5′, SOCS3 and CISH . (M: DNA marker; B: blank; PC: positive control; N: normal control; C1: HEL; C2: MEG-01; C3: SET-2; C4: K562).
    Figure Legend Snippet: Methylation-specific polymerase chain reaction of SOCS1 , SOCS2 -5′, SOCS3, CISH and SHP1 in cell lines. For SHP1 , K562 was homozygously methylated, while HEL, SET-2 and MEG-01 were completely unmethylated. HEL, SET-2, MEG-01 and K562 were completely unmethylated for SOCS1 , SOCS2 -5′, SOCS3 and CISH . (M: DNA marker; B: blank; PC: positive control; N: normal control; C1: HEL; C2: MEG-01; C3: SET-2; C4: K562).

    Techniques Used: Methylation, Polymerase Chain Reaction, Chromogenic In Situ Hybridization, Marker, Positive Control

    Methylation-specific polymerase chain reaction (MSP) of SOCS1 , SOCS2 -5′, SOCS3 , CISH and SHP1 in MPN primary samples. ( A ) M-/U-MSP analysis showed that for SOCS1 , SOCS2 -5′, SOCS3 , CISH , methylation was absent in MPN marrow samples, whereas for SHP1 , methylation was found in four MPN patients. (M: DNA marker; B: blank; PC: positive control; N: normal control; S: primary sample). ( B ) DNA sequencing of SHP1 M-MSP products from bisulphite-converted methylated positive and MPN primary samples showing methylated cytosine [C] residues in CpG dinucleotide remained unchanged, unmethylated C residues were converted into [T], whereas all the non-CpG C residues were unmethylated and were converted to thymidine [T].
    Figure Legend Snippet: Methylation-specific polymerase chain reaction (MSP) of SOCS1 , SOCS2 -5′, SOCS3 , CISH and SHP1 in MPN primary samples. ( A ) M-/U-MSP analysis showed that for SOCS1 , SOCS2 -5′, SOCS3 , CISH , methylation was absent in MPN marrow samples, whereas for SHP1 , methylation was found in four MPN patients. (M: DNA marker; B: blank; PC: positive control; N: normal control; S: primary sample). ( B ) DNA sequencing of SHP1 M-MSP products from bisulphite-converted methylated positive and MPN primary samples showing methylated cytosine [C] residues in CpG dinucleotide remained unchanged, unmethylated C residues were converted into [T], whereas all the non-CpG C residues were unmethylated and were converted to thymidine [T].

    Techniques Used: Methylation, Polymerase Chain Reaction, Chromogenic In Situ Hybridization, Marker, Positive Control, DNA Sequencing

    ( A ) Methylation-specific PCR of normal controls using SOCS2 -3′ methylation-specific polymerase chain reaction (MSP) primers. M-/U-MSP analysis showed that four of five normal peripheral blood controls (PB1-5) were methylated with the SOCS2 -3′ primers. (M: DNA marker; B: blank; PC: positive control; PB, normal peripheral blood control). ( B ) Sequencing of MSP products using SOCS2 -3′ MSP primers in normal controls and positive control showing methylation signals. Methylated cytosine residues [C] in CpG dinucleotide remained as C, whereas unmethylated cytosine read as [T] after bisulphite conversion. (PC: positive control; PB: normal peripheral blood control).
    Figure Legend Snippet: ( A ) Methylation-specific PCR of normal controls using SOCS2 -3′ methylation-specific polymerase chain reaction (MSP) primers. M-/U-MSP analysis showed that four of five normal peripheral blood controls (PB1-5) were methylated with the SOCS2 -3′ primers. (M: DNA marker; B: blank; PC: positive control; PB, normal peripheral blood control). ( B ) Sequencing of MSP products using SOCS2 -3′ MSP primers in normal controls and positive control showing methylation signals. Methylated cytosine residues [C] in CpG dinucleotide remained as C, whereas unmethylated cytosine read as [T] after bisulphite conversion. (PC: positive control; PB: normal peripheral blood control).

    Techniques Used: Methylation, Polymerase Chain Reaction, Marker, Positive Control, Sequencing

    Effect of 5-AzadC treatment on K562 cells. (A) M-/U-methylation-specific polymerase chain reaction analysis of SHP1 promoter methylation status showed that 5-AzadC treatment led to progressive demethylation of SHP1 promoter in K562 cells. (M: DNA marker; B: blank; PC: positive control; N: normal control; D0, day 0; D3, day 3 culture in 5-AzadC with 0.5 μM). (B) Reverse transcription–PCR (RT-PCR) analysis of the GAPDH status and SHP1 expression after 5-AzadC treatment. (M: DNA marker; B: blank; N: normal control; NRT: negative control without reverse transcriptase; D0, day 0; D3, day 3 culture in 5-AzadC with 0.5 μM).
    Figure Legend Snippet: Effect of 5-AzadC treatment on K562 cells. (A) M-/U-methylation-specific polymerase chain reaction analysis of SHP1 promoter methylation status showed that 5-AzadC treatment led to progressive demethylation of SHP1 promoter in K562 cells. (M: DNA marker; B: blank; PC: positive control; N: normal control; D0, day 0; D3, day 3 culture in 5-AzadC with 0.5 μM). (B) Reverse transcription–PCR (RT-PCR) analysis of the GAPDH status and SHP1 expression after 5-AzadC treatment. (M: DNA marker; B: blank; N: normal control; NRT: negative control without reverse transcriptase; D0, day 0; D3, day 3 culture in 5-AzadC with 0.5 μM).

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

    Methylation-specific polymerase chain reaction (MSP) of SOCS1 , SOCS2 -5′, SOCS3 , CISH and SHP1 in normal controls. M-/U-MSP analysis showed that three normal peripheral blood controls (PB1-3) and three bone marrow controls (BM1-3) were unmethylated with the SOCS1 , SOCS2 -5′, SOCS3 , CISH and SHP1 primers. (M: DNA marker; B: blank; PC: positive control; PB, normal peripheral blood control; BM: normal bone marrow control).
    Figure Legend Snippet: Methylation-specific polymerase chain reaction (MSP) of SOCS1 , SOCS2 -5′, SOCS3 , CISH and SHP1 in normal controls. M-/U-MSP analysis showed that three normal peripheral blood controls (PB1-3) and three bone marrow controls (BM1-3) were unmethylated with the SOCS1 , SOCS2 -5′, SOCS3 , CISH and SHP1 primers. (M: DNA marker; B: blank; PC: positive control; PB, normal peripheral blood control; BM: normal bone marrow control).

    Techniques Used: Methylation, Polymerase Chain Reaction, Chromogenic In Situ Hybridization, Marker, Positive Control

    28) Product Images from "Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription"

    Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1149

    Production of HIV-1 DIS RNA mutants from PBMCs partially rescues defects in viral cDNA synthesis. WT and DIS mutant HIV-1 viruses were produced either by transfection of 293T cells or infection of PHA-stimulated freshly isolated PBMCs. Equivalent amounts of DNase treated virus were used to infect either SupT1 cells ( A ) or PBMCs ( B ). Cells were lysed 24 h postinfection and HIV-1 cDNA was measured by real-time PCR. HIV-1 DNA copies were normalized by CCR5. Results are reported as a percentage of the WT control and represent means (±SE) of separate experiments.
    Figure Legend Snippet: Production of HIV-1 DIS RNA mutants from PBMCs partially rescues defects in viral cDNA synthesis. WT and DIS mutant HIV-1 viruses were produced either by transfection of 293T cells or infection of PHA-stimulated freshly isolated PBMCs. Equivalent amounts of DNase treated virus were used to infect either SupT1 cells ( A ) or PBMCs ( B ). Cells were lysed 24 h postinfection and HIV-1 cDNA was measured by real-time PCR. HIV-1 DNA copies were normalized by CCR5. Results are reported as a percentage of the WT control and represent means (±SE) of separate experiments.

    Techniques Used: Mutagenesis, Produced, Transfection, Infection, Isolation, Real-time Polymerase Chain Reaction

    29) Product Images from "Sarcocystis cruzi: First Molecular Identification from Cattle in Iran"

    Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran

    Journal: International Journal of Molecular and Cellular Medicine

    doi:

    Electrophoresis of PCR product of Sarcocystis isolated from goat and calf in northern Iran. Lanes: 1 2, Sarcocystis isolated from goat; 3, Sarcocystis isolated from calf muscle sample; M, 100 bp ladder DNA size marker; and N, negative control
    Figure Legend Snippet: Electrophoresis of PCR product of Sarcocystis isolated from goat and calf in northern Iran. Lanes: 1 2, Sarcocystis isolated from goat; 3, Sarcocystis isolated from calf muscle sample; M, 100 bp ladder DNA size marker; and N, negative control

    Techniques Used: Electrophoresis, Polymerase Chain Reaction, Isolation, Northern Blot, Marker, Negative Control

    30) Product Images from "Genetic Variants in Cytosolic 5?-Nucleotidase II Are Associated with Its Expression and Cytarabine Sensitivity in HapMap Cell Lines and in Patients with Acute Myeloid Leukemia S⃞"

    Article Title: Genetic Variants in Cytosolic 5?-Nucleotidase II Are Associated with Its Expression and Cytarabine Sensitivity in HapMap Cell Lines and in Patients with Acute Myeloid Leukemia S⃞

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.111.182873

    A, NT5C2 promoter with CpG sites. Bisulfite-modified genomic DNA sequence of the CpG region of the human NT5C2 gene. The position of the primers that were used for methylation-specific PCR after bisulfite treatment are indicated by forward and reverse
    Figure Legend Snippet: A, NT5C2 promoter with CpG sites. Bisulfite-modified genomic DNA sequence of the CpG region of the human NT5C2 gene. The position of the primers that were used for methylation-specific PCR after bisulfite treatment are indicated by forward and reverse

    Techniques Used: Modification, Sequencing, Methylation, Polymerase Chain Reaction

    31) Product Images from "Fratricide activity of MafB protein of N. meningitidis strain B16B6"

    Article Title: Fratricide activity of MafB protein of N. meningitidis strain B16B6

    Journal: BMC Microbiology

    doi: 10.1186/s12866-015-0493-6

    Genetic organization of the MGI of strain B16B6. B16B6 contains three MGIs. The genetic composition of each island and their flanking regions (indicated by solid green arrows) are in accordance with those observed in other genomes (Fig. 1 ); therefore, the same classification is applied. The different genomic features of each island are illustrated as indicated in the legend to Fig. 1 . Color coding of genes is also similar as in Fig. 1 . The organization of the MGI of strain B16B6 is compared with those of FAM18, which is of the same clonal complex cc11; similarities are indicated with grey shadowing as in Fig. 1 . MGI-3 of FAM18 is similar to that of B16B6 and is therefore not depicted. DNA fragments targeted by PCR for the generation of knockout constructs are indicated by lines and numbered a-h; the corresponding primers are presented in Additional file 1 : Table S1
    Figure Legend Snippet: Genetic organization of the MGI of strain B16B6. B16B6 contains three MGIs. The genetic composition of each island and their flanking regions (indicated by solid green arrows) are in accordance with those observed in other genomes (Fig. 1 ); therefore, the same classification is applied. The different genomic features of each island are illustrated as indicated in the legend to Fig. 1 . Color coding of genes is also similar as in Fig. 1 . The organization of the MGI of strain B16B6 is compared with those of FAM18, which is of the same clonal complex cc11; similarities are indicated with grey shadowing as in Fig. 1 . MGI-3 of FAM18 is similar to that of B16B6 and is therefore not depicted. DNA fragments targeted by PCR for the generation of knockout constructs are indicated by lines and numbered a-h; the corresponding primers are presented in Additional file 1 : Table S1

    Techniques Used: Polymerase Chain Reaction, Knock-Out, Construct

    32) Product Images from "G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter"

    Article Title: G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113955

    Comparison of G4 conformation in different buffers. CD spectra for G4MEST1L (A), G4MEST2 (B), and G4MEST3 (C) in NaPi, 50 mM KCl (solid line) or PCR buffer (dashed line). (D) CD spectra for G4MEST3 in NaPi, 50 mM KCl (solid line) or NaPi containing 50 mM KCl and 1.5 mM Mg 2+ (dashed line). Molar ellipticity (x10 5 deg.cm 2 .dmol −1 ) is on the vertical axis and wavelength (nm) is on the horizontal axis.
    Figure Legend Snippet: Comparison of G4 conformation in different buffers. CD spectra for G4MEST1L (A), G4MEST2 (B), and G4MEST3 (C) in NaPi, 50 mM KCl (solid line) or PCR buffer (dashed line). (D) CD spectra for G4MEST3 in NaPi, 50 mM KCl (solid line) or NaPi containing 50 mM KCl and 1.5 mM Mg 2+ (dashed line). Molar ellipticity (x10 5 deg.cm 2 .dmol −1 ) is on the vertical axis and wavelength (nm) is on the horizontal axis.

    Techniques Used: Polymerase Chain Reaction

    33) Product Images from "Optimization of a 12-Hour TaqMan PCR-Based Method for Detection of Salmonella Bacteria in Meat † Bacteria in Meat † ▿"

    Article Title: Optimization of a 12-Hour TaqMan PCR-Based Method for Detection of Salmonella Bacteria in Meat † Bacteria in Meat † ▿

    Journal:

    doi: 10.1128/AEM.02823-06

    Amplification plot (FAM) showing the difference in amplification curves obtained by analysis of 5, 10, and 20 μl of template DNA in the PCR from samples of minced pork meat inoculated with 1 to 10 or 10 to 100 CFU of S. enterica serovar Typhimurium
    Figure Legend Snippet: Amplification plot (FAM) showing the difference in amplification curves obtained by analysis of 5, 10, and 20 μl of template DNA in the PCR from samples of minced pork meat inoculated with 1 to 10 or 10 to 100 CFU of S. enterica serovar Typhimurium

    Techniques Used: Amplification, Polymerase Chain Reaction

    34) Product Images from "Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin-proteasome pathway"

    Article Title: Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin-proteasome pathway

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601184

    Effect of treatment of C 2 C 12 myotubes with 15( S )-HETE for 4 h on expression of mRNA for ( A ) C2 proteasome subunit, ( B ) C5 proteasome subunit, ( C ) E2 14k , as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m. from three separate determinations performed on different occasions with different samples. Differences are indicated as (a) P
    Figure Legend Snippet: Effect of treatment of C 2 C 12 myotubes with 15( S )-HETE for 4 h on expression of mRNA for ( A ) C2 proteasome subunit, ( B ) C5 proteasome subunit, ( C ) E2 14k , as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m. from three separate determinations performed on different occasions with different samples. Differences are indicated as (a) P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    35) Product Images from "Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways"

    Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.186.12.3882-3888.2004

    Disruption of the dspA gene. (A) PCR analysis of the dspA gene in potential mutants in which the dspA gene was insertionally inactivated with the cat gene. WT, wild type; M, DNA size marker. (B and C) Depiction of the constructs used to generate the dspA-cat strain (B) and the nblS + dspA mutant strain (C). To generate the dspA-cat disruption, an internal 29-bp dspA fragment from the dspA gene (from Sp to Bm; shaded area) was deleted and replaced by a 0.85-kbp chloramphenicol resistance cartridge (Cat); in panel C, the cat gene shown in panel B was replaced by a 2.2-kb nblS gene linked with a 1.2-kb kanamycin resistance cartridge (Kan); the nblS and kan r genes are transcribed in opposite directions. In panel A, both the intact dspA (∼2.0 kbp) and the dspA-cat (∼2.8 kbp) loci were efficiently amplified from genomic DNA preparations by PCR. A total of 5 chloramphenicol-resistant transformants were isolated (lanes 1 to 5) and analyzed for a disruption of the dspA gene; transformants 3 and 4 showed no detectable wild-type dspA PCR product, and transformant 4 was used in all subsequent analyses. Sp, SphI; Bg, BglII; Bm, BamHI; Xm, XmnI; Sm, SmaI; Pt, PstI.
    Figure Legend Snippet: Disruption of the dspA gene. (A) PCR analysis of the dspA gene in potential mutants in which the dspA gene was insertionally inactivated with the cat gene. WT, wild type; M, DNA size marker. (B and C) Depiction of the constructs used to generate the dspA-cat strain (B) and the nblS + dspA mutant strain (C). To generate the dspA-cat disruption, an internal 29-bp dspA fragment from the dspA gene (from Sp to Bm; shaded area) was deleted and replaced by a 0.85-kbp chloramphenicol resistance cartridge (Cat); in panel C, the cat gene shown in panel B was replaced by a 2.2-kb nblS gene linked with a 1.2-kb kanamycin resistance cartridge (Kan); the nblS and kan r genes are transcribed in opposite directions. In panel A, both the intact dspA (∼2.0 kbp) and the dspA-cat (∼2.8 kbp) loci were efficiently amplified from genomic DNA preparations by PCR. A total of 5 chloramphenicol-resistant transformants were isolated (lanes 1 to 5) and analyzed for a disruption of the dspA gene; transformants 3 and 4 showed no detectable wild-type dspA PCR product, and transformant 4 was used in all subsequent analyses. Sp, SphI; Bg, BglII; Bm, BamHI; Xm, XmnI; Sm, SmaI; Pt, PstI.

    Techniques Used: Polymerase Chain Reaction, Marker, Construct, Mutagenesis, Amplification, Isolation

    36) Product Images from "Evaluation of the Invader Assay, a Linear Signal Amplification Method, for Identification of Mutations Associated with Resistance to Rifampin and Isoniazid in Mycobacterium tuberculosis"

    Article Title: Evaluation of the Invader Assay, a Linear Signal Amplification Method, for Identification of Mutations Associated with Resistance to Rifampin and Isoniazid in Mycobacterium tuberculosis

    Journal: Antimicrobial Agents and Chemotherapy

    doi:

    Identification of Asp 516 →Val mutation in the rpoB gene by the Invader linear signal amplification assay. A wild-type (lanes 1 and 3) or mutant (lanes 2 and 4) target or an equimolar mixture of PCR targets (lanes 5, 6, and 7) was tested against a wild-type (13-nt [lanes 1 and 5]) or mutant (10-nt [lanes 2 and 6]) FAM-labeled probe or against both (lanes 3, 4, and 7). Controls representing the signal probes in various combinations without target are shown in lanes 8, 9, and 10. (A) Samples were diluted 50-fold, and 2 μl was loaded onto a 24% 17-cm denaturing polyacrylamide gel and run on an ABI model 377 sequencing apparatus. Molecular weight markers of 4, 6, 8, 10, 13, and 15 nucleotides were FAM labeled. Data were collected using filter set C processed with GeneScan software. (B) samples (5 μl) were electrophoresed at 20 W on a 20% denaturing polyacrylamide gel and scanned at 505 nm using a Hitachi model FMBIO-100 fluorescence scanner. Molecular weight markers of 4, 6, 8, and 15 nucleotides were TET labeled. nt, nucleotide; wt, wild type; mt, mutant.
    Figure Legend Snippet: Identification of Asp 516 →Val mutation in the rpoB gene by the Invader linear signal amplification assay. A wild-type (lanes 1 and 3) or mutant (lanes 2 and 4) target or an equimolar mixture of PCR targets (lanes 5, 6, and 7) was tested against a wild-type (13-nt [lanes 1 and 5]) or mutant (10-nt [lanes 2 and 6]) FAM-labeled probe or against both (lanes 3, 4, and 7). Controls representing the signal probes in various combinations without target are shown in lanes 8, 9, and 10. (A) Samples were diluted 50-fold, and 2 μl was loaded onto a 24% 17-cm denaturing polyacrylamide gel and run on an ABI model 377 sequencing apparatus. Molecular weight markers of 4, 6, 8, 10, 13, and 15 nucleotides were FAM labeled. Data were collected using filter set C processed with GeneScan software. (B) samples (5 μl) were electrophoresed at 20 W on a 20% denaturing polyacrylamide gel and scanned at 505 nm using a Hitachi model FMBIO-100 fluorescence scanner. Molecular weight markers of 4, 6, 8, and 15 nucleotides were TET labeled. nt, nucleotide; wt, wild type; mt, mutant.

    Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction, Labeling, Sequencing, Molecular Weight, Software, Fluorescence

    37) Product Images from "Long-term health in recipients of transplanted in vitro propagated spermatogonial stem cells"

    Article Title: Long-term health in recipients of transplanted in vitro propagated spermatogonial stem cells

    Journal: Human Reproduction (Oxford, England)

    doi: 10.1093/humrep/dex348

    eGFP is expressed in transplanted testis, but not in benign and malignant lesions outside of the testis. Gel electrophoresis of a standard PCR for eGFP performed on genomic DNA from adult testis and lesions found in transplanted animals. Genomic DNA of adult B6D2F1 actin-eGFP testis serves as a positive control. Mouse 541570 serves as an example for all transplanted mice, and was transplanted unilaterally with eGFP positive GS cells, which can be confirmed by PCR 14 months after transplantation with a positive eGFP signal in the transplanted testis and no signal in the contralateral untransplanted testis. Mouse 541555 developed a hematoma in the transplanted testis, explaining a positive read-out in this case. eGFP could not be detected in all other benign and malignant lesions. An amplicon for β-actin is present in all samples. LN, lymph node; LCL, diffuse large cell lymphoma.
    Figure Legend Snippet: eGFP is expressed in transplanted testis, but not in benign and malignant lesions outside of the testis. Gel electrophoresis of a standard PCR for eGFP performed on genomic DNA from adult testis and lesions found in transplanted animals. Genomic DNA of adult B6D2F1 actin-eGFP testis serves as a positive control. Mouse 541570 serves as an example for all transplanted mice, and was transplanted unilaterally with eGFP positive GS cells, which can be confirmed by PCR 14 months after transplantation with a positive eGFP signal in the transplanted testis and no signal in the contralateral untransplanted testis. Mouse 541555 developed a hematoma in the transplanted testis, explaining a positive read-out in this case. eGFP could not be detected in all other benign and malignant lesions. An amplicon for β-actin is present in all samples. LN, lymph node; LCL, diffuse large cell lymphoma.

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Positive Control, Mouse Assay, Transplantation Assay, Amplification

    38) Product Images from "G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter"

    Article Title: G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113955

    Comparison of G4 conformation in different buffers. CD spectra for G4MEST1L (A), G4MEST2 (B), and G4MEST3 (C) in NaPi, 50 mM KCl (solid line) or PCR buffer (dashed line). (D) CD spectra for G4MEST3 in NaPi, 50 mM KCl (solid line) or NaPi containing 50 mM KCl and 1.5 mM Mg 2+ (dashed line). Molar ellipticity (x10 5 deg.cm 2 .dmol −1 ) is on the vertical axis and wavelength (nm) is on the horizontal axis.
    Figure Legend Snippet: Comparison of G4 conformation in different buffers. CD spectra for G4MEST1L (A), G4MEST2 (B), and G4MEST3 (C) in NaPi, 50 mM KCl (solid line) or PCR buffer (dashed line). (D) CD spectra for G4MEST3 in NaPi, 50 mM KCl (solid line) or NaPi containing 50 mM KCl and 1.5 mM Mg 2+ (dashed line). Molar ellipticity (x10 5 deg.cm 2 .dmol −1 ) is on the vertical axis and wavelength (nm) is on the horizontal axis.

    Techniques Used: Polymerase Chain Reaction

    Comparison of methylated G4 conformation in different buffers. CD spectra for methylated oligonucleotides G4MEST1LM (A), G4MEST2M (B), and G4MEST3M (C) in NaPi, 50 mM KCl (solid line) or PCR buffer (dashed line). Molar ellipticity (x10 5 deg.cm 2 .dmol −1 ) is on the vertical axis and wavelength (nm) is on the horizontal axis.
    Figure Legend Snippet: Comparison of methylated G4 conformation in different buffers. CD spectra for methylated oligonucleotides G4MEST1LM (A), G4MEST2M (B), and G4MEST3M (C) in NaPi, 50 mM KCl (solid line) or PCR buffer (dashed line). Molar ellipticity (x10 5 deg.cm 2 .dmol −1 ) is on the vertical axis and wavelength (nm) is on the horizontal axis.

    Techniques Used: Methylation, Polymerase Chain Reaction

    Synthetic MEST template mixing experiments using marked templates. PCR with primers MESTPF1/MESTPR3C or MESTPF1A/MESTPR3C on the two gBlock constructs (wild-type and mutant) generated synthetic templates that were identical except for the presence of one variant base introduced with the mismatch primer MESTPF1A. Methylated and unmethylated forms of these amplicons were diluted and mixed, subjected to PCR, and then genotyped by Sanger sequencing. Products derived from the synthetic templates could be distinguished due to the presence of either an A or T at this position. (A) wild-type templates for which the “T” allele was methylated, and the “A” allele was unmethylated, showing apparent “A” homozygosity; (B) mutant (non-G4 forming) templates for which the “T” allele was methylated, and the “A” allele was unmethylated, showing apparent heterozygosity (W).
    Figure Legend Snippet: Synthetic MEST template mixing experiments using marked templates. PCR with primers MESTPF1/MESTPR3C or MESTPF1A/MESTPR3C on the two gBlock constructs (wild-type and mutant) generated synthetic templates that were identical except for the presence of one variant base introduced with the mismatch primer MESTPF1A. Methylated and unmethylated forms of these amplicons were diluted and mixed, subjected to PCR, and then genotyped by Sanger sequencing. Products derived from the synthetic templates could be distinguished due to the presence of either an A or T at this position. (A) wild-type templates for which the “T” allele was methylated, and the “A” allele was unmethylated, showing apparent “A” homozygosity; (B) mutant (non-G4 forming) templates for which the “T” allele was methylated, and the “A” allele was unmethylated, showing apparent heterozygosity (W).

    Techniques Used: Polymerase Chain Reaction, Construct, Mutagenesis, Generated, Variant Assay, Methylation, Sequencing, Derivative Assay

    39) Product Images from "Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India"

    Article Title: Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India

    Journal: Journal of Mid-Life Health

    doi: 10.4103/0976-7800.127786

    Human papillomavirus (HPV)18 type-specific polymerase chain reaction products. Lane 7: 50 bp ladder; lanes 1-2 : HPV18 (+) ve samples showing specific band size at 100 bp; lanes 3-5: HPV18 (–)ve sample; lane 6: Positive control; lane 8: Negative control
    Figure Legend Snippet: Human papillomavirus (HPV)18 type-specific polymerase chain reaction products. Lane 7: 50 bp ladder; lanes 1-2 : HPV18 (+) ve samples showing specific band size at 100 bp; lanes 3-5: HPV18 (–)ve sample; lane 6: Positive control; lane 8: Negative control

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

    40) Product Images from "Construction of a System for the Strawberry Nursery Production towards Elimination of Latent Infection of Anthracnose Fungi by a Combination of PCR and Microtube Hybridization"

    Article Title: Construction of a System for the Strawberry Nursery Production towards Elimination of Latent Infection of Anthracnose Fungi by a Combination of PCR and Microtube Hybridization

    Journal: The Plant Pathology Journal

    doi: 10.5423/PPJ.NT.05.2016.0132

    Schematic flow of the diagnostic method developed in this study for latent infection of strawberry anthracnose fungi. The diagnostic steps consist of 4 steps, initial culturing of candidate fungi (Step 1), DNA extraction from fungal hyphae (Step 2), synthesis of PCR-amplified probes (Step 3), and microtube hybridization (MTH) and colorimetric detection (Step 4). The experimental details are described in the text.
    Figure Legend Snippet: Schematic flow of the diagnostic method developed in this study for latent infection of strawberry anthracnose fungi. The diagnostic steps consist of 4 steps, initial culturing of candidate fungi (Step 1), DNA extraction from fungal hyphae (Step 2), synthesis of PCR-amplified probes (Step 3), and microtube hybridization (MTH) and colorimetric detection (Step 4). The experimental details are described in the text.

    Techniques Used: Flow Cytometry, Diagnostic Assay, Infection, DNA Extraction, Polymerase Chain Reaction, Amplification, Hybridization

    Related Articles

    Amplification:

    Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
    Article Snippet: .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany). .. The following conditions were used for amplification: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 53°C for 1 min and extension at 72°C for 1 min and further extension for 5 min at 72°C ( ).

    Synthesized:

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation
    Article Snippet: .. PCR was performed in a 50-μl reaction mixture containing 2 μl of synthesized cDNA product or 0.5 μg of genomic DNA, 5 μl of 10× PCR buffer, 1.5 mM MgCl2, 0.3 mM dNTP, 1.25 unit of Taq polymerase (Roche, http://www.roche.com ), and 6 μM of each primer. .. The primer sequences that were used for the different mRNAs or DNAs were: unrec 5′-GCTAGGGATAACAGGGTAAT-3′; rec 5′-GAGGGCGAGGGCGATGCC-3′; reverse oligo 5′-TGCACGCTGCCGTCCTCG-3′ (443-bp amplified fragment); β-actin/R 5′-AAAGCCATGCCAATCTCATC-3′; β-actin/L 5′-GATCATTGCTCCTCCTGAGC-3′ (250 bp amplified fragment); GAPDH forward oligonucleotide 5′-TTCACCACCACCATGGAGAAGGCT-3′; reverse 5′-ACAGCCTTGGCAGCACCAGT-3′ (346-bp amplified fragment).

    Labeling:

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
    Article Snippet: .. The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample. .. The cycle profile was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 58°C for 60 s. Fluorescence measurements were obtained online and analyzed on the ABI-PRISM with the SDS software (version 1.7a; Applied Biosystems) and on the RotorGene with the version 4.6 software (Corbett Research).

    Real-time Polymerase Chain Reaction:

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
    Article Snippet: .. The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample. .. The cycle profile was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 58°C for 60 s. Fluorescence measurements were obtained online and analyzed on the ABI-PRISM with the SDS software (version 1.7a; Applied Biosystems) and on the RotorGene with the version 4.6 software (Corbett Research).

    Concentration Assay:

    Article Title: Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor
    Article Snippet: .. The DNA sample and primer mixture was complemented with an optimal concentration of PCR Buffer containing 2 mM MgCl2 , PCR grade deoxyribonucleoside triphosphates / dNTP mix (10 mM of each nucleotide), 2U of FastStart Taq DNA Polymerase (Roche, Germany), and nuclease free water. .. Amplification was performed with an Applied Biosystems® Veriti® 96-Well Thermal Cycler (USA).

    Polymerase Chain Reaction:

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation
    Article Snippet: .. PCR was performed in a 50-μl reaction mixture containing 2 μl of synthesized cDNA product or 0.5 μg of genomic DNA, 5 μl of 10× PCR buffer, 1.5 mM MgCl2, 0.3 mM dNTP, 1.25 unit of Taq polymerase (Roche, http://www.roche.com ), and 6 μM of each primer. .. The primer sequences that were used for the different mRNAs or DNAs were: unrec 5′-GCTAGGGATAACAGGGTAAT-3′; rec 5′-GAGGGCGAGGGCGATGCC-3′; reverse oligo 5′-TGCACGCTGCCGTCCTCG-3′ (443-bp amplified fragment); β-actin/R 5′-AAAGCCATGCCAATCTCATC-3′; β-actin/L 5′-GATCATTGCTCCTCCTGAGC-3′ (250 bp amplified fragment); GAPDH forward oligonucleotide 5′-TTCACCACCACCATGGAGAAGGCT-3′; reverse 5′-ACAGCCTTGGCAGCACCAGT-3′ (346-bp amplified fragment).

    Article Title: Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor
    Article Snippet: .. The DNA sample and primer mixture was complemented with an optimal concentration of PCR Buffer containing 2 mM MgCl2 , PCR grade deoxyribonucleoside triphosphates / dNTP mix (10 mM of each nucleotide), 2U of FastStart Taq DNA Polymerase (Roche, Germany), and nuclease free water. .. Amplification was performed with an Applied Biosystems® Veriti® 96-Well Thermal Cycler (USA).

    Article Title: CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1
    Article Snippet: .. Five μg of total RNA were reverse transcribed using 10× PCR buffer (Roche), 10 mM dNTPs, Oligo (dT), M-MLV-RT (all from Invitrogen), and RNAsin (Promega). ..

    Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
    Article Snippet: .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany). .. The following conditions were used for amplification: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 53°C for 1 min and extension at 72°C for 1 min and further extension for 5 min at 72°C ( ).

    Article Title: Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India
    Article Snippet: .. HPV TS-PCR The HPV16-specific PCR mixture (20 μl) contained ×10 PCR buffer, 2 mM MgCl2 , 200 μM dNTP, 4 ng primers, 0.5 U FS Taq and 100 ng DNA. .. The touch-down PCR-program consisted of initial 14 cycles of denaturation (95°C; 30 s), annealing (61°C; 30 s; reduction by 0.5°C/cycle) and elongation (72°C; 30 s), followed by 23 cycles of denaturation (95°C; 30 s), annealing (55°C; 30 s) and elongation (72°C; 30 s), along with initial denaturation and final elongation as above.

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
    Article Snippet: .. The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample. .. The cycle profile was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 58°C for 60 s. Fluorescence measurements were obtained online and analyzed on the ABI-PRISM with the SDS software (version 1.7a; Applied Biosystems) and on the RotorGene with the version 4.6 software (Corbett Research).

    Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
    Article Snippet: .. MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl. ..

    Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents
    Article Snippet: .. PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche). .. PCR conditions were an initial denaturation of 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 52–64 °C for 30 s, and 72 °C for 30 s. PCR products were purified with the Qiaquick PCR purification kit (Qiagen) and sequenced on both strands using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems).

    CTG Assay:

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
    Article Snippet: .. The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample. .. The cycle profile was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 58°C for 60 s. Fluorescence measurements were obtained online and analyzed on the ABI-PRISM with the SDS software (version 1.7a; Applied Biosystems) and on the RotorGene with the version 4.6 software (Corbett Research).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
    Article Snippet: .. The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample. .. The cycle profile was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 58°C for 60 s. Fluorescence measurements were obtained online and analyzed on the ABI-PRISM with the SDS software (version 1.7a; Applied Biosystems) and on the RotorGene with the version 4.6 software (Corbett Research).

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    Roche pcr buffer
    Human papillomavirus (HPV)16 type-specific polymerase chain reaction products. Lane 4: 50 bp ladder; lanes 3, 5-8: <t>HPV16</t> (+) ve samples showing specific band size at 116 bp; lane 9: HPV16 (–)ve sample; lane 2: Positive control; lane 1: Negative control
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    Human papillomavirus (HPV)16 type-specific polymerase chain reaction products. Lane 4: 50 bp ladder; lanes 3, 5-8: HPV16 (+) ve samples showing specific band size at 116 bp; lane 9: HPV16 (–)ve sample; lane 2: Positive control; lane 1: Negative control

    Journal: Journal of Mid-Life Health

    Article Title: Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India

    doi: 10.4103/0976-7800.127786

    Figure Lengend Snippet: Human papillomavirus (HPV)16 type-specific polymerase chain reaction products. Lane 4: 50 bp ladder; lanes 3, 5-8: HPV16 (+) ve samples showing specific band size at 116 bp; lane 9: HPV16 (–)ve sample; lane 2: Positive control; lane 1: Negative control

    Article Snippet: HPV TS-PCR The HPV16-specific PCR mixture (20 μl) contained ×10 PCR buffer, 2 mM MgCl2 , 200 μM dNTP, 4 ng primers, 0.5 U FS Taq and 100 ng DNA.

    Techniques: Polymerase Chain Reaction, Positive Control, Negative Control

    Flow chart depicting study design. TS-PCR: Type-specific polymerase chain reaction, HPV: Human papillomavirus, H and E: Hematoxylin and eosin, PAS: Periodic Acid-Schiff

    Journal: Journal of Mid-Life Health

    Article Title: Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India

    doi: 10.4103/0976-7800.127786

    Figure Lengend Snippet: Flow chart depicting study design. TS-PCR: Type-specific polymerase chain reaction, HPV: Human papillomavirus, H and E: Hematoxylin and eosin, PAS: Periodic Acid-Schiff

    Article Snippet: HPV TS-PCR The HPV16-specific PCR mixture (20 μl) contained ×10 PCR buffer, 2 mM MgCl2 , 200 μM dNTP, 4 ng primers, 0.5 U FS Taq and 100 ng DNA.

    Techniques: Flow Cytometry, Polymerase Chain Reaction

    The linear range of the real-time PCR method when detecting purified DNA from C. jejuni CCUG 11284 on the RotorGene 3000 (5 × 10 1 to 1 × 10 7 copies; □) and the ABI-PRISM 7700 (10 3 to 10 7 copies; ▴). A real-time PCR sample

    Journal: Applied and Environmental Microbiology

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters

    doi: 10.1128/AEM.70.6.3588-3592.2004

    Figure Lengend Snippet: The linear range of the real-time PCR method when detecting purified DNA from C. jejuni CCUG 11284 on the RotorGene 3000 (5 × 10 1 to 1 × 10 7 copies; □) and the ABI-PRISM 7700 (10 3 to 10 7 copies; ▴). A real-time PCR sample

    Article Snippet: The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample.

    Techniques: Real-time Polymerase Chain Reaction, Purification

    Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

    Journal: Clinical Epigenetics

    Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents

    doi: 10.1186/s13148-018-0594-9

    Figure Lengend Snippet: Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

    Article Snippet: PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche).

    Techniques: Methylation, Mass Spectrometry, Polymerase Chain Reaction, Derivative Assay, Positive Control, Molecular Weight

    v-miRs modulate expression of cellular miRNAs in human oral keratinocytes (HOK). Heat maps showing differentially expressed cellular miRNAs in (A) miR-H1, (B) miR-K12-3-3p, and (C) miR-UL70-3p transfected HOK. (D) Venn diagram showing the distribution of unique and overlapping altered cellular miRNAs in v-miR-transfected HOK. (E) Validation of microarray by quantitative RT-PCR for miR-7, miR-21-3p, and miR-29b (marked by black arrows in the heat map) in another separate cohort of transfected HOK. RNU6 was used as endogenous control. Data are expressed as mean ± SEM of four independent transfections. Student’s t -test was conducted to calculate p -values (* p

    Journal: Frontiers in Immunology

    Article Title: Viral miRNAs Alter Host Cell miRNA Profiles and Modulate Innate Immune Responses

    doi: 10.3389/fimmu.2018.00433

    Figure Lengend Snippet: v-miRs modulate expression of cellular miRNAs in human oral keratinocytes (HOK). Heat maps showing differentially expressed cellular miRNAs in (A) miR-H1, (B) miR-K12-3-3p, and (C) miR-UL70-3p transfected HOK. (D) Venn diagram showing the distribution of unique and overlapping altered cellular miRNAs in v-miR-transfected HOK. (E) Validation of microarray by quantitative RT-PCR for miR-7, miR-21-3p, and miR-29b (marked by black arrows in the heat map) in another separate cohort of transfected HOK. RNU6 was used as endogenous control. Data are expressed as mean ± SEM of four independent transfections. Student’s t -test was conducted to calculate p -values (* p

    Article Snippet: The reactions were run using miRNA specific primer and universal primer (both from Qiagen) in the PCR mix buffer containing SYBR Green (Roche, Indianapolis, IN, USA).

    Techniques: Expressing, Transfection, Microarray, Quantitative RT-PCR