sybr green pcr master mix  (Thermo Fisher)


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    Name:
    SYBR Green PCR Master Mix
    Description:
    Alternative Product Try PowerUp SYBR Green Master Mix our newest high performance SYBR dye based master mix for superior performance at a very competitive price With PowerUp SYBR Green Master Mix we ve taken the best of SYBR Green PCR Master Mix and added additional capabilities for your gene expression analysis Everything you need for SYBR Green dye based PCR amplification and detection in a convenient single tube format Applied Biosystems SYBR Green PCR Master Mix combines SYBR Green I dye AmpliTaq Gold DNA Polymerase dNTPs with dUTP Passive Reference 1 and optimized buffer in the convenience of a single vial • Premixed components stored at 2 8°C significantly reduce assay setup time• SYBR Green I dye detects double stranded DNA so specific probes are not required• AmpliTaq Gold DNA polymerase minimizes nonspecific product formation to achieve superior performance• dUTP significantly reduces carryover contamination when used in conjunction with uracil DNA glycosylase• Proprietary buffer enhancements ensure performance and reliabilityMaximum Flexibility and ConvenienceApplied Biosystems SYBR Green PCR Master Mix provides maximum flexibility at reduced cost because no target specific TaqMan probes are required SYBR Green I dye is a double stranded DNA binding dye that detects any double stranded DNA generated during PCR The hot start enzyme AmpliTaq Gold DNA Polymerase minimizes nonspecific product formation including primer dimers yielding superior performance and sensitivity Passive Internal Reference 1 is provided to normalize non PCR related fluorescence fluctuations This minimizes well to well variability that can result from a variety of causes such as pipetting error or sample evaporation SYBR Green I dye is ideal for target identification screening assays or when a limited number of assays is needed
    Catalog Number:
    4309155
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    Applications:
    Enzymes & Master Mixes for Real-Time PCR|PCR & Real-Time PCR|Real Time PCR (qPCR)|Real Time PCR-Based Gene Expression Profiling|Gene Expression Analysis & Genotyping
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    Kits and Assays
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    Structured Review

    Thermo Fisher sybr green pcr master mix
    Impact of nonspecific signal in <t>PCR</t> quantification read-outs. Individual cells expressing or not Gzmb mRNA were amplified simultaneously. ( A ) <t>SYBR</t> Green signal in positive (solid lines) and negative (dotted lines) cells. ( B ) C T evaluation, using the Sequence Detector 1.7 software. The C T value of negative cells and wells not containing template was not significantly different ( t -test: P = 0.18). This program's upper limit of detection is 60 cycles; that is, samples without template score with a C T of 60.
    Alternative Product Try PowerUp SYBR Green Master Mix our newest high performance SYBR dye based master mix for superior performance at a very competitive price With PowerUp SYBR Green Master Mix we ve taken the best of SYBR Green PCR Master Mix and added additional capabilities for your gene expression analysis Everything you need for SYBR Green dye based PCR amplification and detection in a convenient single tube format Applied Biosystems SYBR Green PCR Master Mix combines SYBR Green I dye AmpliTaq Gold DNA Polymerase dNTPs with dUTP Passive Reference 1 and optimized buffer in the convenience of a single vial • Premixed components stored at 2 8°C significantly reduce assay setup time• SYBR Green I dye detects double stranded DNA so specific probes are not required• AmpliTaq Gold DNA polymerase minimizes nonspecific product formation to achieve superior performance• dUTP significantly reduces carryover contamination when used in conjunction with uracil DNA glycosylase• Proprietary buffer enhancements ensure performance and reliabilityMaximum Flexibility and ConvenienceApplied Biosystems SYBR Green PCR Master Mix provides maximum flexibility at reduced cost because no target specific TaqMan probes are required SYBR Green I dye is a double stranded DNA binding dye that detects any double stranded DNA generated during PCR The hot start enzyme AmpliTaq Gold DNA Polymerase minimizes nonspecific product formation including primer dimers yielding superior performance and sensitivity Passive Internal Reference 1 is provided to normalize non PCR related fluorescence fluctuations This minimizes well to well variability that can result from a variety of causes such as pipetting error or sample evaporation SYBR Green I dye is ideal for target identification screening assays or when a limited number of assays is needed
    https://www.bioz.com/result/sybr green pcr master mix/product/Thermo Fisher
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    Images

    1) Product Images from "Quantification of Multiple Gene Expression in Individual Cells"

    Article Title: Quantification of Multiple Gene Expression in Individual Cells

    Journal: Genome Research

    doi: 10.1101/gr.2890204

    Impact of nonspecific signal in PCR quantification read-outs. Individual cells expressing or not Gzmb mRNA were amplified simultaneously. ( A ) SYBR Green signal in positive (solid lines) and negative (dotted lines) cells. ( B ) C T evaluation, using the Sequence Detector 1.7 software. The C T value of negative cells and wells not containing template was not significantly different ( t -test: P = 0.18). This program's upper limit of detection is 60 cycles; that is, samples without template score with a C T of 60.
    Figure Legend Snippet: Impact of nonspecific signal in PCR quantification read-outs. Individual cells expressing or not Gzmb mRNA were amplified simultaneously. ( A ) SYBR Green signal in positive (solid lines) and negative (dotted lines) cells. ( B ) C T evaluation, using the Sequence Detector 1.7 software. The C T value of negative cells and wells not containing template was not significantly different ( t -test: P = 0.18). This program's upper limit of detection is 60 cycles; that is, samples without template score with a C T of 60.

    Techniques Used: Polymerase Chain Reaction, Expressing, Amplification, SYBR Green Assay, Sequencing, Software

    2) Product Images from "CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use"

    Article Title: CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-6-5

    Comparison to TaqMan ® technology. Total RNA was extracted from isolated monocytes stimulated for 6 hours by LPS, serial-diluted 1:10 and retro-transcribed to generate standard curves by plotting the C T against the concentration of this cellular RNA in arbitrary units. TNF-α transcripts were amplified by real time PCR using either ( A ) the TaqMan ® commercial kit and the TaqMan ® universal PCR master mix or ( B ) our primers and the SYBR Green PCR master mix. Data represent the standard curves obtained for the two techniques, their slopes and the deducted efficiencies.
    Figure Legend Snippet: Comparison to TaqMan ® technology. Total RNA was extracted from isolated monocytes stimulated for 6 hours by LPS, serial-diluted 1:10 and retro-transcribed to generate standard curves by plotting the C T against the concentration of this cellular RNA in arbitrary units. TNF-α transcripts were amplified by real time PCR using either ( A ) the TaqMan ® commercial kit and the TaqMan ® universal PCR master mix or ( B ) our primers and the SYBR Green PCR master mix. Data represent the standard curves obtained for the two techniques, their slopes and the deducted efficiencies.

    Techniques Used: Isolation, Concentration Assay, Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, SYBR Green Assay

    Primer validation. A. Agarose gel electrophoresis of several CyProQuant-PCR products generated by amplification of a pool of cDNA from PBMC stimulated in vitro by LPS or PHA. Scale is shown in base pairs (bp). B. Dissociation curve analysis of these CyProQuant-PCR products. Negative derivative of the fluorescence is plotted against temperature. The single peak shows that SYBR Green fluorescence detects only the specific CyProQuant-PCR product. C. Amplification plots and standard curve resulting from the amplification of a range of β2-MG external RNA standard using CyProQuant-PCR.
    Figure Legend Snippet: Primer validation. A. Agarose gel electrophoresis of several CyProQuant-PCR products generated by amplification of a pool of cDNA from PBMC stimulated in vitro by LPS or PHA. Scale is shown in base pairs (bp). B. Dissociation curve analysis of these CyProQuant-PCR products. Negative derivative of the fluorescence is plotted against temperature. The single peak shows that SYBR Green fluorescence detects only the specific CyProQuant-PCR product. C. Amplification plots and standard curve resulting from the amplification of a range of β2-MG external RNA standard using CyProQuant-PCR.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Generated, Amplification, In Vitro, Fluorescence, SYBR Green Assay

    3) Product Images from "hCAF1/CNOT7 regulates interferon signalling by targeting STAT1"

    Article Title: hCAF1/CNOT7 regulates interferon signalling by targeting STAT1

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2013.11

    Characterization of hCAF1-depleted cells. MCF7 cells were transfected with vectors expressing control miRNA (mock) or with two alternative miRNAs (called kd, and kd-1) targeting hCAF1. After vector transfection and selection, mock, hCAF1 kd and hCAF1 kd-1 cells were obtained. Total RNA or protein extracts were prepared to test hCAF1 knockdown efficiency. ( A ) Left panel, SYBR green real-time RT–PCR analysis was performed for detection of transcript levels of hCAF1. Results were normalized using 36B4 mRNA level as an internal control. Transcript levels in control cells (mock) were set to 1. Right panel, hCAF1 protein levels were analysed by western blot from 30 μg of protein extracts. ( B ) Endogenous hCAF1 expression and localization in hCAF1 kd and hCAF1 kd-1 compared to the mock control, by Immunofluorescent staining using mouse polyclonal anti-CAF1 antibody and goat anti-mouse Alexa 488-conjugated secondary antibody (green). Scale bar=20 μm. ( C ) Upper: summary of Human Exon 1.0 ST Array analysis results. Diagram of the hierarchical clustering of gene expression profiles of control versus hCAF1-deficient hCAF1 kd cell lines. Probes are represented vertically, whereas conditions are shown horizontally. Lower: type I and II interferon distribution by ISG Database. ( D ) Validation of the DNA array screen. SYBR green RT-qPCR analysis of the upregulated gene products IFI27, IFI6, TAP1, STAT1, IFITM1, HERC6, PLSCR1 and the downregulated gene CSTA. Total RNA isolated from mock and hCAF1 kd cells was reverse transcribed, and PCR was performed with primers specific for the transcripts of the indicated genes. Gene expression levels were normalized to internal controls 36B4 and shown as expression levels of hCAF1 kd cells relative to expression levels in control cells (arbitrarily set to 1). ( E , F ) Rescue of hCAF1 functions in knockdown cells. hCAF1 kd cells were stably transfected with a plasmid expressing mouse flag CAF1 (insensitive to miRNAs), or with the empty vector used as a control. ( E ) The expression of the indicated genes was analysed by real-time RT-qPCR as in ( D ). ( F ) Efficiency of flag mCAF1 overexpression and the protein expression of the indicated hCAF1-regulated genes was assessed by western blot. The experiments illustrated in ( D ) and ( E ) were performed in triplicate and expressed as mean values of three independent experiments. Standard deviations are shown. page.
    Figure Legend Snippet: Characterization of hCAF1-depleted cells. MCF7 cells were transfected with vectors expressing control miRNA (mock) or with two alternative miRNAs (called kd, and kd-1) targeting hCAF1. After vector transfection and selection, mock, hCAF1 kd and hCAF1 kd-1 cells were obtained. Total RNA or protein extracts were prepared to test hCAF1 knockdown efficiency. ( A ) Left panel, SYBR green real-time RT–PCR analysis was performed for detection of transcript levels of hCAF1. Results were normalized using 36B4 mRNA level as an internal control. Transcript levels in control cells (mock) were set to 1. Right panel, hCAF1 protein levels were analysed by western blot from 30 μg of protein extracts. ( B ) Endogenous hCAF1 expression and localization in hCAF1 kd and hCAF1 kd-1 compared to the mock control, by Immunofluorescent staining using mouse polyclonal anti-CAF1 antibody and goat anti-mouse Alexa 488-conjugated secondary antibody (green). Scale bar=20 μm. ( C ) Upper: summary of Human Exon 1.0 ST Array analysis results. Diagram of the hierarchical clustering of gene expression profiles of control versus hCAF1-deficient hCAF1 kd cell lines. Probes are represented vertically, whereas conditions are shown horizontally. Lower: type I and II interferon distribution by ISG Database. ( D ) Validation of the DNA array screen. SYBR green RT-qPCR analysis of the upregulated gene products IFI27, IFI6, TAP1, STAT1, IFITM1, HERC6, PLSCR1 and the downregulated gene CSTA. Total RNA isolated from mock and hCAF1 kd cells was reverse transcribed, and PCR was performed with primers specific for the transcripts of the indicated genes. Gene expression levels were normalized to internal controls 36B4 and shown as expression levels of hCAF1 kd cells relative to expression levels in control cells (arbitrarily set to 1). ( E , F ) Rescue of hCAF1 functions in knockdown cells. hCAF1 kd cells were stably transfected with a plasmid expressing mouse flag CAF1 (insensitive to miRNAs), or with the empty vector used as a control. ( E ) The expression of the indicated genes was analysed by real-time RT-qPCR as in ( D ). ( F ) Efficiency of flag mCAF1 overexpression and the protein expression of the indicated hCAF1-regulated genes was assessed by western blot. The experiments illustrated in ( D ) and ( E ) were performed in triplicate and expressed as mean values of three independent experiments. Standard deviations are shown. page.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Selection, SYBR Green Assay, Quantitative RT-PCR, Western Blot, Staining, DNA Array, Isolation, Polymerase Chain Reaction, Stable Transfection, Over Expression, Polyacrylamide Gel Electrophoresis

    4) Product Images from "BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection"

    Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection

    Journal: Virology Journal

    doi: 10.1186/s12985-015-0266-8

    Curcumin decreases BRCA1 occupancy at the HIV-1 LTR. A . TZM-bl cells were transfected with pcTat and treated the next day with vehicle (DMSO) or a titration of curcumin (0.5, 1, 10, and 20 μM). Samples were analyzed by western blot. Inset depicts a dose–response curve of BRCA1 protein abundance versus curcumin treatment based on the average densitometry counts with error bars representing standard error of three independent measurements. Western blot is representative of three independent experiments. Densitometry counts were taken from three independent treatments to acquire a dose–response curve of BRCA1 expression inhibition (inset plot) B . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or 20 μM curcumin. Bright-Glo luciferase assays and CellTiter-Glo cell viability assays were performed 24 hours post-treatment as described by the manufacturer. Data was normalized to cells containing Tat and treated with DMSO as baseline for Tat-dependent LTR activation. Transfection and treatment assays were performed in triplicate and data plotted represents averaged data of two independent experiments. Error bars show the standard error of two averaged independent measurements. Viability assays were performed in triplicate. C . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or curcumin (20 μM) for 24 hours prior to being collected for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-IgG (10 μg), and anti-RNA polymerase II (RNAP II, 10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. Single asterisk indicates p
    Figure Legend Snippet: Curcumin decreases BRCA1 occupancy at the HIV-1 LTR. A . TZM-bl cells were transfected with pcTat and treated the next day with vehicle (DMSO) or a titration of curcumin (0.5, 1, 10, and 20 μM). Samples were analyzed by western blot. Inset depicts a dose–response curve of BRCA1 protein abundance versus curcumin treatment based on the average densitometry counts with error bars representing standard error of three independent measurements. Western blot is representative of three independent experiments. Densitometry counts were taken from three independent treatments to acquire a dose–response curve of BRCA1 expression inhibition (inset plot) B . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or 20 μM curcumin. Bright-Glo luciferase assays and CellTiter-Glo cell viability assays were performed 24 hours post-treatment as described by the manufacturer. Data was normalized to cells containing Tat and treated with DMSO as baseline for Tat-dependent LTR activation. Transfection and treatment assays were performed in triplicate and data plotted represents averaged data of two independent experiments. Error bars show the standard error of two averaged independent measurements. Viability assays were performed in triplicate. C . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or curcumin (20 μM) for 24 hours prior to being collected for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-IgG (10 μg), and anti-RNA polymerase II (RNAP II, 10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. Single asterisk indicates p

    Techniques Used: Transfection, Titration, Western Blot, Expressing, Inhibition, Luciferase, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction, Immunoprecipitation

    BRCA1 is present at the HIV-1 LTR in HIV infected T-cells. A . CEM cells were infected with NL4-3 virus (p24 = 5000 pg/ml) for 4 hours and collected 72 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-V5 (10 μg), and anti-histone H3-phosphorylated at S10 (pS10-H3, 5 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. B . CEM cells were pre-treated with DMSO or 10 μM ATM inhibitor (ATM in ) for 2 hours. Cells were then infected with NL4-3 virus (p24 = 5000 pg/ml) for 4 hours, followed by post-treating the cells with DMSO or ATM in . Cells were collected 72 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), p-BRCA1 S1423 (10 μg), and anti-V5 (10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. Double asterisk indicates statistically significant difference p ≤ 0.01.
    Figure Legend Snippet: BRCA1 is present at the HIV-1 LTR in HIV infected T-cells. A . CEM cells were infected with NL4-3 virus (p24 = 5000 pg/ml) for 4 hours and collected 72 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-V5 (10 μg), and anti-histone H3-phosphorylated at S10 (pS10-H3, 5 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. B . CEM cells were pre-treated with DMSO or 10 μM ATM inhibitor (ATM in ) for 2 hours. Cells were then infected with NL4-3 virus (p24 = 5000 pg/ml) for 4 hours, followed by post-treating the cells with DMSO or ATM in . Cells were collected 72 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), p-BRCA1 S1423 (10 μg), and anti-V5 (10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. Double asterisk indicates statistically significant difference p ≤ 0.01.

    Techniques Used: Infection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction, Immunoprecipitation

    5) Product Images from "A Myc-microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes"

    Article Title: A Myc-microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1452

    Myc activates miR-22 during the transition from quiescence to proliferation. ( A ) Heat map showing the response of miRNAs to Myc overexpression (OE), and Myc knockdown (KO) in HeLa cells, and to serum stimulation of quiescent fibroblasts (SS) measured using microarrays. Ratios displayed under the SS column represent the maximum activation across all measured time points (5, 10, 20, 30, 60 and 180 min) after serum stimulation. miR-17 and miR-92 are previously known downstream targets of Myc. ( B ) miR-22 expression is inhibited by Myc knockdown in proliferating fibroblasts. miR-22 expression was assayed by qRT-PCR in fibroblasts transfected with an siRNA against Myc or a control siRNA. Relative expression values were normalized to those obtained using the control siRNA ( Y -axis). ( C ) ChIP-seq data for Myc in HeLaS3, MCF7 and serum-stimulated fibroblasts show Myc binding upstream of miR-22. The profiles of overlapping extended ChIP-seq reads are indicated in green. Chromosome coordinates are indicated on top and genes are shown at bottom. MIR22HG refers to the primary miR-22 transcript as annotated in RefSeq, and the mature miR-22 sequence is indicated by a red symbol. Regions (L1–L4) subsequently tested for promoter activity in luciferase assays are shown by black rectangles, phylogenetically conserved E-boxes by vertical blue lines and CpG islands and qPCR primer loci by vertical black lines. ( D ) ChIP-qPCR verification of Myc binding upstream of miR-22. An independent ChIP for Myc was performed in serum-stimulated fibroblasts. Primers were designed to amplify two loci (F1 and F2) indicated as binding sites by ChIP-seq. Fold enrichment, indicative of Myc binding, was calculated relative to input DNA, normalized to a negative control region ( Y -axis). The threshold for ChIP enrichment relative to the control is indicated by the red dashed line. ( E ) Promoter assays for 4 Myc putative promoter regions upstream of miR-22. L1–L4 on the X -axis indicate cloned candidate promoter regions. P is a positive control. The Y -axis shows expression fold change of a luciferase promoter reporter normalized to a negative control vector. The L3 region shows a significant increase in reporter activity when Myc is overexpressed. ( F ) Mutation of putative Myc-binding sites M1 and M2 in the L3 promoter region. M1 + M2 indicates both sites were mutated. For E and F, gray bars indicate Myc overexpression and white bars indicate endogenous levels of Myc (Myc was not overexpressed). For B, D, E and F, bars indicate the mean, and error bars denote ±SD, n = 3. For B, E and F, * P
    Figure Legend Snippet: Myc activates miR-22 during the transition from quiescence to proliferation. ( A ) Heat map showing the response of miRNAs to Myc overexpression (OE), and Myc knockdown (KO) in HeLa cells, and to serum stimulation of quiescent fibroblasts (SS) measured using microarrays. Ratios displayed under the SS column represent the maximum activation across all measured time points (5, 10, 20, 30, 60 and 180 min) after serum stimulation. miR-17 and miR-92 are previously known downstream targets of Myc. ( B ) miR-22 expression is inhibited by Myc knockdown in proliferating fibroblasts. miR-22 expression was assayed by qRT-PCR in fibroblasts transfected with an siRNA against Myc or a control siRNA. Relative expression values were normalized to those obtained using the control siRNA ( Y -axis). ( C ) ChIP-seq data for Myc in HeLaS3, MCF7 and serum-stimulated fibroblasts show Myc binding upstream of miR-22. The profiles of overlapping extended ChIP-seq reads are indicated in green. Chromosome coordinates are indicated on top and genes are shown at bottom. MIR22HG refers to the primary miR-22 transcript as annotated in RefSeq, and the mature miR-22 sequence is indicated by a red symbol. Regions (L1–L4) subsequently tested for promoter activity in luciferase assays are shown by black rectangles, phylogenetically conserved E-boxes by vertical blue lines and CpG islands and qPCR primer loci by vertical black lines. ( D ) ChIP-qPCR verification of Myc binding upstream of miR-22. An independent ChIP for Myc was performed in serum-stimulated fibroblasts. Primers were designed to amplify two loci (F1 and F2) indicated as binding sites by ChIP-seq. Fold enrichment, indicative of Myc binding, was calculated relative to input DNA, normalized to a negative control region ( Y -axis). The threshold for ChIP enrichment relative to the control is indicated by the red dashed line. ( E ) Promoter assays for 4 Myc putative promoter regions upstream of miR-22. L1–L4 on the X -axis indicate cloned candidate promoter regions. P is a positive control. The Y -axis shows expression fold change of a luciferase promoter reporter normalized to a negative control vector. The L3 region shows a significant increase in reporter activity when Myc is overexpressed. ( F ) Mutation of putative Myc-binding sites M1 and M2 in the L3 promoter region. M1 + M2 indicates both sites were mutated. For E and F, gray bars indicate Myc overexpression and white bars indicate endogenous levels of Myc (Myc was not overexpressed). For B, D, E and F, bars indicate the mean, and error bars denote ±SD, n = 3. For B, E and F, * P

    Techniques Used: Over Expression, Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Chromatin Immunoprecipitation, Binding Assay, Sequencing, Activity Assay, Luciferase, Real-time Polymerase Chain Reaction, Negative Control, Clone Assay, Positive Control, Plasmid Preparation, Mutagenesis

    miR-22 target genes mediating interferon suppression. ( A ) The interferon response pathway. Green indicates genes in the pathway that were repressed by miR-22 in quiescent fibroblasts but have no predicted miR-22 target sites; orange indicates genes repressed by miR-22 in quiescent fibroblasts and have predicted miR-22 target sites; blue indicates genes that have predicted target sites but were not repressed by miR-22 in quiescent fibroblasts. Genes in gray do not meet any of these criteria. ( B ) Ago2 IPs identify HMGB1 and IRF5 as direct miR-22 targets. HMGB1 and IRF5 transcripts show significantly higher Ago2 occupancy in miR-22-transfected HeLa cells compared with mock-transfected cells. Ago2 occupancy of the target genes transcripts was measured using qRT-PCR as described in the text. The Y -axis shows fold change in mRNA levels from Ago2 IP–isolated RNA normalized to input RNA. ( C ) Luciferase assays show that miR-22 directly targets the 3′UTRs of HMGB1 and IRF5. The Y -axis indicates relative luciferase units normalized to the control siRNA transfection. IKKg and Empty are negative controls. IKKg was not repressed by miR-22 and Empty is the luciferase vector with no UTR. For the mutant 3′UTRs, 3 bp in each 6-mer miR-22 target site in the 3′UTRs were mutated. ( D ) IRF5 protein expression in fibroblasts was downregulated by transfection with miR-22 compared with control siRNA transfection. ( E ) Increased expression of IRF5 in quiescent fibroblasts relative to proliferating fibroblasts assayed by qRT-PCR. Y -axis indicates fold enrichment in quiescent versus proliferating fibroblasts. ( F ) IRF5 protein expression was increased in quiescent compared with proliferating fibroblasts. For B and E, GAPDH was used as a control for normalizing input RNA levels. For B, C and E, bars are the mean, and error bars denote ±SD, n = 3. For B, C, and E, P -values were estimated by Student’s t -test. * P
    Figure Legend Snippet: miR-22 target genes mediating interferon suppression. ( A ) The interferon response pathway. Green indicates genes in the pathway that were repressed by miR-22 in quiescent fibroblasts but have no predicted miR-22 target sites; orange indicates genes repressed by miR-22 in quiescent fibroblasts and have predicted miR-22 target sites; blue indicates genes that have predicted target sites but were not repressed by miR-22 in quiescent fibroblasts. Genes in gray do not meet any of these criteria. ( B ) Ago2 IPs identify HMGB1 and IRF5 as direct miR-22 targets. HMGB1 and IRF5 transcripts show significantly higher Ago2 occupancy in miR-22-transfected HeLa cells compared with mock-transfected cells. Ago2 occupancy of the target genes transcripts was measured using qRT-PCR as described in the text. The Y -axis shows fold change in mRNA levels from Ago2 IP–isolated RNA normalized to input RNA. ( C ) Luciferase assays show that miR-22 directly targets the 3′UTRs of HMGB1 and IRF5. The Y -axis indicates relative luciferase units normalized to the control siRNA transfection. IKKg and Empty are negative controls. IKKg was not repressed by miR-22 and Empty is the luciferase vector with no UTR. For the mutant 3′UTRs, 3 bp in each 6-mer miR-22 target site in the 3′UTRs were mutated. ( D ) IRF5 protein expression in fibroblasts was downregulated by transfection with miR-22 compared with control siRNA transfection. ( E ) Increased expression of IRF5 in quiescent fibroblasts relative to proliferating fibroblasts assayed by qRT-PCR. Y -axis indicates fold enrichment in quiescent versus proliferating fibroblasts. ( F ) IRF5 protein expression was increased in quiescent compared with proliferating fibroblasts. For B and E, GAPDH was used as a control for normalizing input RNA levels. For B, C and E, bars are the mean, and error bars denote ±SD, n = 3. For B, C, and E, P -values were estimated by Student’s t -test. * P

    Techniques Used: Transfection, Quantitative RT-PCR, Isolation, Luciferase, Plasmid Preparation, Mutagenesis, Expressing

    6) Product Images from "RNase L restricts the mobility of engineered retrotransposons in cultured human cells"

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1308

    RNase L reduces L1 RNA accumulation in cells. ( A ) Results of qRT-PCR experiments: HeLa-M cells were co-transfected with pAD2TE1 and an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. L1 RNA levels were determined 48 h after transfection using the Sybr Green method ( 84 ). The X-axis indicates the RNase L co-transfected samples. The Y-axis indicates the relative expression level of L1 RNA from the transfected construct. The L1 RNA amounts were normalized with hygromycin mRNA levels (see ‘Materials and Methods’ section for detailed PCR strategy). Data are represented as the mean ± SD from three technical replicates of a single representative experiment. * P
    Figure Legend Snippet: RNase L reduces L1 RNA accumulation in cells. ( A ) Results of qRT-PCR experiments: HeLa-M cells were co-transfected with pAD2TE1 and an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. L1 RNA levels were determined 48 h after transfection using the Sybr Green method ( 84 ). The X-axis indicates the RNase L co-transfected samples. The Y-axis indicates the relative expression level of L1 RNA from the transfected construct. The L1 RNA amounts were normalized with hygromycin mRNA levels (see ‘Materials and Methods’ section for detailed PCR strategy). Data are represented as the mean ± SD from three technical replicates of a single representative experiment. * P

    Techniques Used: Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, SYBR Green Assay, Construct, Polymerase Chain Reaction

    7) Product Images from "Synergistic effects of snail and quercetin on renal cell carcinoma Caki-2 by altering AKT/mTOR/ERK1/2 signaling pathways"

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

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

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

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

    8) Product Images from "NADPH oxidase expression and production of superoxide by human corneal stromal cells"

    Article Title: NADPH oxidase expression and production of superoxide by human corneal stromal cells

    Journal: Molecular Vision

    doi:

    Relative mRNA content of cells treated with small interfering RNAs to NOX1 , NOX4 , or NOX5 . Human corneal stromal fibroblasts were treated with 40 nM small intefering (si)RNAs to nontarget controls (lanes 1,3, and 5) or NOX1 -specific (lane 2), NOX4 -specific (lane 4), or NOX5 -specific (lane 6) siRNAs. RNA was extracted and amplified in triplicate amplification by real-time reverse transcriptase PCR, using the SYBR Green. Each bar represents the mean and standard error of the mean of triplicate amplifications of RNAs extracted from triplicate cultures. A significant reduction (p
    Figure Legend Snippet: Relative mRNA content of cells treated with small interfering RNAs to NOX1 , NOX4 , or NOX5 . Human corneal stromal fibroblasts were treated with 40 nM small intefering (si)RNAs to nontarget controls (lanes 1,3, and 5) or NOX1 -specific (lane 2), NOX4 -specific (lane 4), or NOX5 -specific (lane 6) siRNAs. RNA was extracted and amplified in triplicate amplification by real-time reverse transcriptase PCR, using the SYBR Green. Each bar represents the mean and standard error of the mean of triplicate amplifications of RNAs extracted from triplicate cultures. A significant reduction (p

    Techniques Used: Amplification, Polymerase Chain Reaction, SYBR Green Assay

    9) Product Images from "The mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility"

    Article Title: The mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility

    Journal: Molecular Immunology

    doi: 10.1016/j.molimm.2007.05.011

    Semi-quantitative RT-PCR to estimate relative expression of CD59a and CD59b in testis (A) and perfused liver (C). Different primer pairs for each isoform and different dilutions of the cDNA-templates (numbers above) were used. Thirty-five cycles were carried out and reaction products were separated in a 1% agarose gel. The average densitometric intensity for each band calculated from three independent experiments is given. Panels B and D present band intensity for CD59a (♦) and CD59b (■) as function of template dilution for testis and liver, respectively. (E) SYBR Green QPCR analysis of expression of CD59b in liver without and with perfusion using primers specific for CD59b. Number of the CD59b mRNA copies in liver (set as 1 for unperfused) was compared that in testis. Data are mean of two independent experiments ± S.E.M.
    Figure Legend Snippet: Semi-quantitative RT-PCR to estimate relative expression of CD59a and CD59b in testis (A) and perfused liver (C). Different primer pairs for each isoform and different dilutions of the cDNA-templates (numbers above) were used. Thirty-five cycles were carried out and reaction products were separated in a 1% agarose gel. The average densitometric intensity for each band calculated from three independent experiments is given. Panels B and D present band intensity for CD59a (♦) and CD59b (■) as function of template dilution for testis and liver, respectively. (E) SYBR Green QPCR analysis of expression of CD59b in liver without and with perfusion using primers specific for CD59b. Number of the CD59b mRNA copies in liver (set as 1 for unperfused) was compared that in testis. Data are mean of two independent experiments ± S.E.M.

    Techniques Used: Quantitative RT-PCR, Expressing, Agarose Gel Electrophoresis, SYBR Green Assay, Real-time Polymerase Chain Reaction

    10) Product Images from "MicroRNAs Distinguish Cytogenetic Subgroups in Pediatric AML and Contribute to Complex Regulatory Networks in AML-Relevant Pathways"

    Article Title: MicroRNAs Distinguish Cytogenetic Subgroups in Pediatric AML and Contribute to Complex Regulatory Networks in AML-Relevant Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056334

    Ago-associated miRNAs and - mRNAs using the PAR-CLIP-Array method. (A) Western Blot analysis of immunoprecipitates of human Ago1-4 from AML cell lines, KASUMI-1 with t(8;21) and NB4 carrying t(15;17). The immunoprecipitates show a specific band of the Argonaute protein (∼97 kDa; ←) in contrast to the isotype control antibody (rat IgG2a) and empty-bead control. A representative sample of the biological triplicate is shown. Please note that more material was loaded for Ago3 and Ago4 since these two Ago proteins are much lower expressed as was also validated by qRT-PCR (not shown). Antibodies were tested for specificity for detection of native and denatured protein prior to this experiment with cell lines overexpressing tagged Ago protein (not shown) (B) Validation of miRNA- and mRNA-enrichment in immunoprecipitation experiments. Argonaute proteins (black bar) are compared to the isotype (white bar) and empty bead controls (grey bar) using TaqMan qRT-PCR assays for microRNA- (upper panel) and SYBR Green qRT-PCR assays for mRNA-quantification (lower panel). Shown are the measured levels (2 −C T) of six and five miRNAs of KASUMI-1 cells (upper left panel) and NB4 cells (upper right panel), respectively. Immunoprecipitation experiments as well as cDNA synthesis were each done in triplicates and the mean value of the nine values as well as one standard deviation is depicted. miRNAs differentially expressed in patient samples between the t(8;21) and t(15;17) were selected together with ubiquitously expressed miR-16. Please note that calculation of ΔC T -values is not possible due to the lack of a housekeeping gene bound to Argonaute proteins. Six Ago-associated mRNAs in the KASUMI-1 cells (lower left panel) and NB4 cells (lower right panel), covering the whole range from low to high enrichment over isotype control according to microarray data, were selected for qRT-PCR validation. Graphs are centered around a C T -value of 29.9 cycles (2 −C T = 0 −9 ).
    Figure Legend Snippet: Ago-associated miRNAs and - mRNAs using the PAR-CLIP-Array method. (A) Western Blot analysis of immunoprecipitates of human Ago1-4 from AML cell lines, KASUMI-1 with t(8;21) and NB4 carrying t(15;17). The immunoprecipitates show a specific band of the Argonaute protein (∼97 kDa; ←) in contrast to the isotype control antibody (rat IgG2a) and empty-bead control. A representative sample of the biological triplicate is shown. Please note that more material was loaded for Ago3 and Ago4 since these two Ago proteins are much lower expressed as was also validated by qRT-PCR (not shown). Antibodies were tested for specificity for detection of native and denatured protein prior to this experiment with cell lines overexpressing tagged Ago protein (not shown) (B) Validation of miRNA- and mRNA-enrichment in immunoprecipitation experiments. Argonaute proteins (black bar) are compared to the isotype (white bar) and empty bead controls (grey bar) using TaqMan qRT-PCR assays for microRNA- (upper panel) and SYBR Green qRT-PCR assays for mRNA-quantification (lower panel). Shown are the measured levels (2 −C T) of six and five miRNAs of KASUMI-1 cells (upper left panel) and NB4 cells (upper right panel), respectively. Immunoprecipitation experiments as well as cDNA synthesis were each done in triplicates and the mean value of the nine values as well as one standard deviation is depicted. miRNAs differentially expressed in patient samples between the t(8;21) and t(15;17) were selected together with ubiquitously expressed miR-16. Please note that calculation of ΔC T -values is not possible due to the lack of a housekeeping gene bound to Argonaute proteins. Six Ago-associated mRNAs in the KASUMI-1 cells (lower left panel) and NB4 cells (lower right panel), covering the whole range from low to high enrichment over isotype control according to microarray data, were selected for qRT-PCR validation. Graphs are centered around a C T -value of 29.9 cycles (2 −C T = 0 −9 ).

    Techniques Used: Cross-linking Immunoprecipitation, Western Blot, Quantitative RT-PCR, Immunoprecipitation, SYBR Green Assay, Standard Deviation, Microarray

    11) Product Images from "Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism"

    Article Title: Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026751

    GW3965 alters dexamethasone-induced mRNA expression of its responsive genes in rat liver in microarray analysis. A, B and C: GW3965 modulates the transcriptional activity of a fraction of dexamethasone-responsive genes. Venn diagrams demonstrating the number of genes regulated by dexamethasone (Dex) and/or GW3965 (A), Dex and/or Dex+GW3965 (B) and GW3965 and/or Dex+GW3965 (C). The common biologic pathways regulated independently by Dex or GW3965 are also demonstrated in the right side of panel A. D. The 72-hour GW3965 treatment does not alter GR protein levels in the rat liver. Tissue lysates were prepared from rat livers treated with indicated compounds. GR ( upper panel ) and β-actin ( lower panel ) were visualized with their specific antibodies in Western blots. E. The number of genes regulated by Dex and GW3965 in respective biologic pathways is shown. Nomenclatures of the demonstrated biologic activities are based on the ontology of the Gene Ontology: http://www.geneontology.org . F. GW3965 does not influence mRNA expression of dexamethasone-suppressive genes in rat livers. Total RNA was harvested from rat livers and mRNA levels of Ccl9 ( left upper panel ), Cxcl12 ( right upper panel ) and H6pd ( lower panel ) were measured with the SYBR-Green real-time PCR. Bars represent mean ± S.E. values of the fold induction of mRNA expression. *: p
    Figure Legend Snippet: GW3965 alters dexamethasone-induced mRNA expression of its responsive genes in rat liver in microarray analysis. A, B and C: GW3965 modulates the transcriptional activity of a fraction of dexamethasone-responsive genes. Venn diagrams demonstrating the number of genes regulated by dexamethasone (Dex) and/or GW3965 (A), Dex and/or Dex+GW3965 (B) and GW3965 and/or Dex+GW3965 (C). The common biologic pathways regulated independently by Dex or GW3965 are also demonstrated in the right side of panel A. D. The 72-hour GW3965 treatment does not alter GR protein levels in the rat liver. Tissue lysates were prepared from rat livers treated with indicated compounds. GR ( upper panel ) and β-actin ( lower panel ) were visualized with their specific antibodies in Western blots. E. The number of genes regulated by Dex and GW3965 in respective biologic pathways is shown. Nomenclatures of the demonstrated biologic activities are based on the ontology of the Gene Ontology: http://www.geneontology.org . F. GW3965 does not influence mRNA expression of dexamethasone-suppressive genes in rat livers. Total RNA was harvested from rat livers and mRNA levels of Ccl9 ( left upper panel ), Cxcl12 ( right upper panel ) and H6pd ( lower panel ) were measured with the SYBR-Green real-time PCR. Bars represent mean ± S.E. values of the fold induction of mRNA expression. *: p

    Techniques Used: Expressing, Microarray, Activity Assay, Western Blot, SYBR Green Assay, Real-time Polymerase Chain Reaction

    Ligand activation of endogenous LXRs differentially regulates dexamethasone-induced mRNA expression of glucocorticoid-responsive genes in a gene-specific fashion. A, B, C, D, E, F and G. HepG2 cells were transfected with negative control siRNA for luciferase GL2 (white bars) or siRNAs for LXRα/β (black bars), and were treated with or without 10 −6 M dexamethasone and/or 10 −6 M GW3965. Total RNA was harvested and mRNA levels of G6Pase (A), PEPCK (B), GILZ (C), ABCG1 (D), LXRα (E), LXRβ (F) and GR (G) were measured with the SYBR-Green real-time PCR. Bars represent mean ± S.E. values of the fold induction of mRNA expression. *: p
    Figure Legend Snippet: Ligand activation of endogenous LXRs differentially regulates dexamethasone-induced mRNA expression of glucocorticoid-responsive genes in a gene-specific fashion. A, B, C, D, E, F and G. HepG2 cells were transfected with negative control siRNA for luciferase GL2 (white bars) or siRNAs for LXRα/β (black bars), and were treated with or without 10 −6 M dexamethasone and/or 10 −6 M GW3965. Total RNA was harvested and mRNA levels of G6Pase (A), PEPCK (B), GILZ (C), ABCG1 (D), LXRα (E), LXRβ (F) and GR (G) were measured with the SYBR-Green real-time PCR. Bars represent mean ± S.E. values of the fold induction of mRNA expression. *: p

    Techniques Used: Activation Assay, Expressing, Transfection, Negative Control, Luciferase, SYBR Green Assay, Real-time Polymerase Chain Reaction

    12) Product Images from "Specific PKC isoforms regulate LPS-stimulated iNOS induction in murine microglial cells"

    Article Title: Specific PKC isoforms regulate LPS-stimulated iNOS induction in murine microglial cells

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-8-38

    Downregulation of PKC isoforms by specific PKC siRNA . A , BV-2 cells transfected with DY-547 labeled RISC-free siRNA in 24-well plate were photographed using a Nikon Eclipse TE2000-U. The white field shows total cells, TRITC represents positively transfected cells, and merge stands for overlaid images. B , BV-2 cells were transfected with individual PKC isoform-specific siRNAs for 48 hr. Total RNAs were isolated from each transfected group for analysis of gene expression by reverse transcriptase PCR using primer pairs specific for PKC δ, ε, θ, η, α, β and GAPDH. The products were run on a 1% agarose gel impregnated with ethidium bromide. The bands were visualized under UV light. In each transfection panel, starting from the left, the lanes stand for DNA marker, RISC-free control siRNA and specific PKC siRNA, respectively. GAPDH was used as a loading control. C , Total mRNA was extracted from BV-2 cells that were transfected with PKC isoform siRNA or RISC free control siRNA (CON) for 48 hr and subjected to cDNA synthesis. The correspondent cDNA was then used as template for quantitative real time PCR with SYBR Green PCR Master mix. ***, p
    Figure Legend Snippet: Downregulation of PKC isoforms by specific PKC siRNA . A , BV-2 cells transfected with DY-547 labeled RISC-free siRNA in 24-well plate were photographed using a Nikon Eclipse TE2000-U. The white field shows total cells, TRITC represents positively transfected cells, and merge stands for overlaid images. B , BV-2 cells were transfected with individual PKC isoform-specific siRNAs for 48 hr. Total RNAs were isolated from each transfected group for analysis of gene expression by reverse transcriptase PCR using primer pairs specific for PKC δ, ε, θ, η, α, β and GAPDH. The products were run on a 1% agarose gel impregnated with ethidium bromide. The bands were visualized under UV light. In each transfection panel, starting from the left, the lanes stand for DNA marker, RISC-free control siRNA and specific PKC siRNA, respectively. GAPDH was used as a loading control. C , Total mRNA was extracted from BV-2 cells that were transfected with PKC isoform siRNA or RISC free control siRNA (CON) for 48 hr and subjected to cDNA synthesis. The correspondent cDNA was then used as template for quantitative real time PCR with SYBR Green PCR Master mix. ***, p

    Techniques Used: Transfection, Labeling, Isolation, Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Real-time Polymerase Chain Reaction, SYBR Green Assay

    13) Product Images from "A Novel S100A8/A9 Induced Fingerprint of Mesenchymal Stem Cells associated with Enhanced Wound Healing"

    Article Title: A Novel S100A8/A9 Induced Fingerprint of Mesenchymal Stem Cells associated with Enhanced Wound Healing

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24425-9

    Validation of RNA-seq data by qRT-PCR. Total RNA and miRNA was extracted from control MSCs and from MSCs which have been treated with 0.5 µg/mL S100A8/A9. Isolated RNAs were assessed for quality with Bioanalyzer and reverse-transcribed using the Reverse Transcription System. The primers targeted (a) CCR7, (b) IL-32, (c) ICOSLG, (d) PRSS36, (e) SERPINA9, (f) PI15, ( g ) MMP27, (h) SPOCK2, ( i ) DSC2, ( j ) hsa-miR-582-5p, to amplify cDNA using 2x power SYBR Green Mix (Applied Biosystems). Relative mRNA or miR levels were calculated by normalizing to β-actin and RNU6B in case of miRNA. Box plots are expressed as mean with min-max wiskers representing the normalized expression level of each gene of MSCs treated with S100A8/A9 as opposed to control MSCs. Data were analyzed using the unpaired, two-tailed non-parametric Student’s t test using Wilcoxon signed rank-test and the differential expression of the qPCR results were expressed as log 2 fold change with the respective p-values. P-value
    Figure Legend Snippet: Validation of RNA-seq data by qRT-PCR. Total RNA and miRNA was extracted from control MSCs and from MSCs which have been treated with 0.5 µg/mL S100A8/A9. Isolated RNAs were assessed for quality with Bioanalyzer and reverse-transcribed using the Reverse Transcription System. The primers targeted (a) CCR7, (b) IL-32, (c) ICOSLG, (d) PRSS36, (e) SERPINA9, (f) PI15, ( g ) MMP27, (h) SPOCK2, ( i ) DSC2, ( j ) hsa-miR-582-5p, to amplify cDNA using 2x power SYBR Green Mix (Applied Biosystems). Relative mRNA or miR levels were calculated by normalizing to β-actin and RNU6B in case of miRNA. Box plots are expressed as mean with min-max wiskers representing the normalized expression level of each gene of MSCs treated with S100A8/A9 as opposed to control MSCs. Data were analyzed using the unpaired, two-tailed non-parametric Student’s t test using Wilcoxon signed rank-test and the differential expression of the qPCR results were expressed as log 2 fold change with the respective p-values. P-value

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Isolation, SYBR Green Assay, Expressing, Two Tailed Test, Real-time Polymerase Chain Reaction

    14) Product Images from "Increased Tumor Oxygenation and Drug Uptake During Anti-Angiogenic Weekly Low Dose Cyclophosphamide Enhances the Anti-Tumor Effect of Weekly Tirapazamine"

    Article Title: Increased Tumor Oxygenation and Drug Uptake During Anti-Angiogenic Weekly Low Dose Cyclophosphamide Enhances the Anti-Tumor Effect of Weekly Tirapazamine

    Journal: Current cancer drug targets

    doi:

    Expression profiles of factors involved in tumor angiogenesis during metronomic chemotherapy. Relative levels of rat VEGF, TSP-I, PLGF, HIF-1α, and mouse VEGF receptors 1 and 2 in 9L tumors were quantified by real-time PCR (qPCR) using SYBR Green
    Figure Legend Snippet: Expression profiles of factors involved in tumor angiogenesis during metronomic chemotherapy. Relative levels of rat VEGF, TSP-I, PLGF, HIF-1α, and mouse VEGF receptors 1 and 2 in 9L tumors were quantified by real-time PCR (qPCR) using SYBR Green

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

    15) Product Images from "Intestine-specific transcription factor Cdx2 induces E-cadherin function by enhancing the trafficking of E-cadherin to the cell membrane"

    Article Title: Intestine-specific transcription factor Cdx2 induces E-cadherin function by enhancing the trafficking of E-cadherin to the cell membrane

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00297.2010

    Cdx2 regulates RTK activity and adherens junction function by inducing caveolin-1 mRNA and protein in Colo 205 cells. A : quantitative SYBR Green RT-PCR analysis of caveolin-1 gene expression in Colo 205 cells. Total RNA was isolated from Colo-MIGR-Cdx2
    Figure Legend Snippet: Cdx2 regulates RTK activity and adherens junction function by inducing caveolin-1 mRNA and protein in Colo 205 cells. A : quantitative SYBR Green RT-PCR analysis of caveolin-1 gene expression in Colo 205 cells. Total RNA was isolated from Colo-MIGR-Cdx2

    Techniques Used: Activity Assay, SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation

    16) Product Images from "Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis"

    Article Title: Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001021

    Chromatin immunoprecipitation (ChIP) reveals co-occupancy at the promoters of Cic target genes by Atxn1 and Cic. (A) ChIP using Cic antisera confirmed Cic binding at the promoter of two direct targets of Capicua, Ccnd1 and Etv5 , that were up-regulated in the Atxn1 −/− and Atxn1 154Q/+ cerebella. (B) ChIP using Atxn1 anti-sera in cerebellar extracts from Atxn1 +/− , Atxn1 154Q/− , and Atxn1 −/− mice reveals a signal in mice expressing only wild-type ( Atxn1 +/− ) but not polyQ-Atxn1 ( Atxn1 154Q/− ) compared to negative controls (pre-immune sera, and Atxn1 −/− ) (C) ChIP as in (B), this time using Cic antibody. In contrast to (B), Cic is present at comparable levels at the target promoters in Atxn1 +/− , Atxn1 154Q/− , and Atxn −/− mice. All ChIP assays were repeated three times on independent samples, representative results shown. (D) ChIP followed by quantitative PCR (ChIP-qPCR) on the promoter of Etv5 confirms that the proportion of immunoprecipitated DNA by Cic antibody is comparable in all three genotypes (as seen in C). A region containing two Capicua binding sites (promoter-CBS) of Etv5 is more enriched by Cic antibody than a region lacking CBSs (promoter-no CBS) compared to preimmune sera. (E) ChIP-qPCR on the promoter of Ccnd1 also shows similar enrichment of immunoprecipitated DNA by Cic antibody in all three genotypes (as seen in C). ChIP-qPCR using primers designed for a region within 100 bps of the CBS in the Ccnd1 promoter (which is highly conserved across species) show more Cic binding than primers designed for a poorly conserved region further downstream (∼400 bps) of the CBS at the promoter of Ccnd1 , compared to preimmune sera. ChIP-qPCR experiments in (D) and (E) were performed in triplicate on three independent sets of samples (3 cerebella per genotype) using SYBR Green Dye. N.S. = not significant, ** p
    Figure Legend Snippet: Chromatin immunoprecipitation (ChIP) reveals co-occupancy at the promoters of Cic target genes by Atxn1 and Cic. (A) ChIP using Cic antisera confirmed Cic binding at the promoter of two direct targets of Capicua, Ccnd1 and Etv5 , that were up-regulated in the Atxn1 −/− and Atxn1 154Q/+ cerebella. (B) ChIP using Atxn1 anti-sera in cerebellar extracts from Atxn1 +/− , Atxn1 154Q/− , and Atxn1 −/− mice reveals a signal in mice expressing only wild-type ( Atxn1 +/− ) but not polyQ-Atxn1 ( Atxn1 154Q/− ) compared to negative controls (pre-immune sera, and Atxn1 −/− ) (C) ChIP as in (B), this time using Cic antibody. In contrast to (B), Cic is present at comparable levels at the target promoters in Atxn1 +/− , Atxn1 154Q/− , and Atxn −/− mice. All ChIP assays were repeated three times on independent samples, representative results shown. (D) ChIP followed by quantitative PCR (ChIP-qPCR) on the promoter of Etv5 confirms that the proportion of immunoprecipitated DNA by Cic antibody is comparable in all three genotypes (as seen in C). A region containing two Capicua binding sites (promoter-CBS) of Etv5 is more enriched by Cic antibody than a region lacking CBSs (promoter-no CBS) compared to preimmune sera. (E) ChIP-qPCR on the promoter of Ccnd1 also shows similar enrichment of immunoprecipitated DNA by Cic antibody in all three genotypes (as seen in C). ChIP-qPCR using primers designed for a region within 100 bps of the CBS in the Ccnd1 promoter (which is highly conserved across species) show more Cic binding than primers designed for a poorly conserved region further downstream (∼400 bps) of the CBS at the promoter of Ccnd1 , compared to preimmune sera. ChIP-qPCR experiments in (D) and (E) were performed in triplicate on three independent sets of samples (3 cerebella per genotype) using SYBR Green Dye. N.S. = not significant, ** p

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

    17) Product Images from "Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes"

    Article Title: Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-6-36

    Tm analysis with SYBR ® Green I of 101-bp PCR products to discriminate the H/R alleles. Panel A: Melting profile without DMSO. Panel B: Melting profile with DMSO 5%. Panel C: Melting profile with DMSO 10%. Heterozygotes are depicted as thick lines, the heterozygous pattern is distinguishable from the two homozygous curves because of the shoulder, most likely due to the presence of heteroduplexes.
    Figure Legend Snippet: Tm analysis with SYBR ® Green I of 101-bp PCR products to discriminate the H/R alleles. Panel A: Melting profile without DMSO. Panel B: Melting profile with DMSO 5%. Panel C: Melting profile with DMSO 10%. Heterozygotes are depicted as thick lines, the heterozygous pattern is distinguishable from the two homozygous curves because of the shoulder, most likely due to the presence of heteroduplexes.

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction

    18) Product Images from "Age-related macular degeneration-associated variants at chromosome 10q26 do not significantly alter ARMS2 and HTRA1 transcript levels in the human retina"

    Article Title: Age-related macular degeneration-associated variants at chromosome 10q26 do not significantly alter ARMS2 and HTRA1 transcript levels in the human retina

    Journal: Molecular Vision

    doi:

    Relationship of ARMS2 and HTRA1 expression in the human placenta to the genotypes at 10q26. We show the effect of AMD-associated variants near ARMS2 and HTRA1 genes on ARMS2 and HTRA1 expression in human placenta samples. A - D : The fold change in expression of the target genes ( ARMS2, HTRA1, HPRT1 , and GAPDH ) relative to the internal control gene ( rRNA ) were correlated to genotypes of corresponding placenta samples. Real-time qRT–PCR reactions were performed in quadruplicate with Taqman gene expression or SYBR Green assays. The fold change average in the expression of the target gene was calculated using the 2 -ddCt method; ddCt=(C t ,Target - C t ,rRNA) experiment - (C t ,Target - C t ,rRNA) reference . Gene expressions in wild-type (having common variants at all four genotyped loci) placentas were used as a reference. cBlack unique symbols (e.g., triangle, square, diamond, dash, X’s cross, circle) indicate individual sample changes, and white square shows the average. Abbreviations: N represents number of samples with given genotypes; P represents polymorphism (P/P represents homozygous; P/- represents heterozygous change. Variants are shown in the left column).
    Figure Legend Snippet: Relationship of ARMS2 and HTRA1 expression in the human placenta to the genotypes at 10q26. We show the effect of AMD-associated variants near ARMS2 and HTRA1 genes on ARMS2 and HTRA1 expression in human placenta samples. A - D : The fold change in expression of the target genes ( ARMS2, HTRA1, HPRT1 , and GAPDH ) relative to the internal control gene ( rRNA ) were correlated to genotypes of corresponding placenta samples. Real-time qRT–PCR reactions were performed in quadruplicate with Taqman gene expression or SYBR Green assays. The fold change average in the expression of the target gene was calculated using the 2 -ddCt method; ddCt=(C t ,Target - C t ,rRNA) experiment - (C t ,Target - C t ,rRNA) reference . Gene expressions in wild-type (having common variants at all four genotyped loci) placentas were used as a reference. cBlack unique symbols (e.g., triangle, square, diamond, dash, X’s cross, circle) indicate individual sample changes, and white square shows the average. Abbreviations: N represents number of samples with given genotypes; P represents polymorphism (P/P represents homozygous; P/- represents heterozygous change. Variants are shown in the left column).

    Techniques Used: Expressing, Quantitative RT-PCR, SYBR Green Assay

    Relationship of ARMS2 and HTRA1 expression in the human retina to the genotypes at 10q26. A : Schematic representation of positions of polymorphic variants at chromosome 10q26. White boxes indicate exons and gray shows translated regions. Numbers indicate the nucleotide position in NT_030059.12 (NCBI Reference Sequence). B - E : Gene expression in the human retina. We investigated the effect of AMD-associated variants near ARMS2 and HTRA1 genes on expression levels of ARMS2 and HTRA1 in human retina samples. Fold changes in expression of the target genes ( ARMS2 , HTRA1 , HPRT1 , and GAPDH ) relative to the internal control gene ( rRNA ) were correlated to the genotypes of four polymorphisms at 10q26. The samples were analyzed after qRT–PCR reactions performed in quadruplicate with Taqman gene expression or SYBR Green assays. The fold change average in expression of the target gene was calculated using the 2 -ddCt method; ddCt=(C t ,Target - C t ,rRNA) experiment - (C t ,Target - C t ,rRNA) reference . Gene expressions in wild-type (with common variants at the four loci) older normal retinas were used as a reference. Black unique symbols (e.g., triangle, square, diamond, dash, X’s cross, circle) indicate individual sample changes, and white square shows the average. Abbreviations: Avg. represents average; N represents number of samples with a given genotype; P represents polymorphism (P/P represents homozygous; P/- represents heterozygous change. Variants are in the left column).
    Figure Legend Snippet: Relationship of ARMS2 and HTRA1 expression in the human retina to the genotypes at 10q26. A : Schematic representation of positions of polymorphic variants at chromosome 10q26. White boxes indicate exons and gray shows translated regions. Numbers indicate the nucleotide position in NT_030059.12 (NCBI Reference Sequence). B - E : Gene expression in the human retina. We investigated the effect of AMD-associated variants near ARMS2 and HTRA1 genes on expression levels of ARMS2 and HTRA1 in human retina samples. Fold changes in expression of the target genes ( ARMS2 , HTRA1 , HPRT1 , and GAPDH ) relative to the internal control gene ( rRNA ) were correlated to the genotypes of four polymorphisms at 10q26. The samples were analyzed after qRT–PCR reactions performed in quadruplicate with Taqman gene expression or SYBR Green assays. The fold change average in expression of the target gene was calculated using the 2 -ddCt method; ddCt=(C t ,Target - C t ,rRNA) experiment - (C t ,Target - C t ,rRNA) reference . Gene expressions in wild-type (with common variants at the four loci) older normal retinas were used as a reference. Black unique symbols (e.g., triangle, square, diamond, dash, X’s cross, circle) indicate individual sample changes, and white square shows the average. Abbreviations: Avg. represents average; N represents number of samples with a given genotype; P represents polymorphism (P/P represents homozygous; P/- represents heterozygous change. Variants are in the left column).

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR, SYBR Green Assay

    19) Product Images from "Corilagin suppresses cholangiocarcinoma progression through Notch signaling pathway in vitro and in vivo"

    Article Title: Corilagin suppresses cholangiocarcinoma progression through Notch signaling pathway in vitro and in vivo

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2016.3413

    Corilagin induces CCA cell apoptosis. (A and B) Two CCA cell lines (QBC939 and MZ-Cha-1) were treated with corilagin at increasing concentrations for 24 h and analyzed for apoptosis by flow cytometry. (C and D) QBC939 and MZ-Cha-1 cells were harvested and RNA was isolated from each group. SYBR green real-time PCR reactions were performed to detect the alterations of bcl-2 (C) and caspase 3 (D) gene expression treated with corilagin for 24 h. Each column represents the mean of three independent experiments. * P
    Figure Legend Snippet: Corilagin induces CCA cell apoptosis. (A and B) Two CCA cell lines (QBC939 and MZ-Cha-1) were treated with corilagin at increasing concentrations for 24 h and analyzed for apoptosis by flow cytometry. (C and D) QBC939 and MZ-Cha-1 cells were harvested and RNA was isolated from each group. SYBR green real-time PCR reactions were performed to detect the alterations of bcl-2 (C) and caspase 3 (D) gene expression treated with corilagin for 24 h. Each column represents the mean of three independent experiments. * P

    Techniques Used: Flow Cytometry, Cytometry, Isolation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Expressing

    20) Product Images from "Validation of SYBR Green based quantification assay for the detection of human Torque Teno virus titers from plasma"

    Article Title: Validation of SYBR Green based quantification assay for the detection of human Torque Teno virus titers from plasma

    Journal: Virology Journal

    doi: 10.1186/1743-422X-10-191

    Agarose gel (3%) electrophoresis of SYBR Green real-time PCR products. The gel picture shows the presence of primer-dimers in negative control (Lane 6 and Lane 7) and specific TTV amplified products (96 bp) in Lanes 2, 3, 4 and 5. Lane 1 and Lane 8 contained 50 bp DNA ladder.
    Figure Legend Snippet: Agarose gel (3%) electrophoresis of SYBR Green real-time PCR products. The gel picture shows the presence of primer-dimers in negative control (Lane 6 and Lane 7) and specific TTV amplified products (96 bp) in Lanes 2, 3, 4 and 5. Lane 1 and Lane 8 contained 50 bp DNA ladder.

    Techniques Used: Agarose Gel Electrophoresis, Electrophoresis, SYBR Green Assay, Real-time Polymerase Chain Reaction, Negative Control, Amplification

    TTV DNA in healthy individuals (n=20) and in HSCT recipients (n=30) by SYBR Green assay. A represents the log copies per mL of plasma for each healthy individual and HSCT recipient tested. B shows the melting curves for HSCT recipients and C shows 3% agarose gel picture with 50 bp DNA ladder in Lane 1, Lane 2 contained positive control (plasmid vector containing TTV insert), Lane 3 - Lane 15 shows the presence of TTV specific PCR products (96 bp) from HSCT recipients while Lane 16 contains the negative control.
    Figure Legend Snippet: TTV DNA in healthy individuals (n=20) and in HSCT recipients (n=30) by SYBR Green assay. A represents the log copies per mL of plasma for each healthy individual and HSCT recipient tested. B shows the melting curves for HSCT recipients and C shows 3% agarose gel picture with 50 bp DNA ladder in Lane 1, Lane 2 contained positive control (plasmid vector containing TTV insert), Lane 3 - Lane 15 shows the presence of TTV specific PCR products (96 bp) from HSCT recipients while Lane 16 contains the negative control.

    Techniques Used: SYBR Green Assay, Agarose Gel Electrophoresis, Positive Control, Plasmid Preparation, Polymerase Chain Reaction, Negative Control

    21) Product Images from "Myocardin Is a Critical Serum Response Factor Cofactor in the Transcriptional Program Regulating Smooth Muscle Cell Differentiation"

    Article Title: Myocardin Is a Critical Serum Response Factor Cofactor in the Transcriptional Program Regulating Smooth Muscle Cell Differentiation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.7.2425-2437.2003

    Forced expression of myocardin induces expression of endogenous SMC genes in undifferentiated mouse ES cells. (A) Forced expression of myocardin in SM22α +/ lacZ ES cells induces transcription of the endogenous SM22α gene. SM22α +/ lacZ ES cells were transfected with the pcDNA3 control plasmid (upper panel) or the pcDNA-Myocardin expression plasmid (lower panel). At 48 h posttransfection, cells were fixed and stained for β-galactosidase activity. β-Galactosidase activity (blue staining) was not observed in the cells transfected with pcDNA3. In contrast, β-galactosidase activity (intense blue staining) was observed in the SM22α +/ lacZ ES cells transfected with pcDNA-Myocardin. (B) Myocardin-induced SMC gene expression in undifferentiated ES cells is SRF dependent. SRF −/− ES cells were transiently transfected with the control plasmid pcDNA3 or expression plasmids encoding myocardin, SRF, or myocardin and SRF. At 48 h posttransfection, the cells were harvested and RNA was prepared. Quantitative real-time PCR was performed with Applied Biosystems SYBR Green PCR Master Mix and MJ Research DNA Engine Opticon 2 real-time detection system. All RT-PCRs were performed in duplicate with (+) and without (−) RT controls. Primer pairs were designed to quantitatively amplify the mouse myocardin, SM22α, SM-α-actin, calponin-h1,
    Figure Legend Snippet: Forced expression of myocardin induces expression of endogenous SMC genes in undifferentiated mouse ES cells. (A) Forced expression of myocardin in SM22α +/ lacZ ES cells induces transcription of the endogenous SM22α gene. SM22α +/ lacZ ES cells were transfected with the pcDNA3 control plasmid (upper panel) or the pcDNA-Myocardin expression plasmid (lower panel). At 48 h posttransfection, cells were fixed and stained for β-galactosidase activity. β-Galactosidase activity (blue staining) was not observed in the cells transfected with pcDNA3. In contrast, β-galactosidase activity (intense blue staining) was observed in the SM22α +/ lacZ ES cells transfected with pcDNA-Myocardin. (B) Myocardin-induced SMC gene expression in undifferentiated ES cells is SRF dependent. SRF −/− ES cells were transiently transfected with the control plasmid pcDNA3 or expression plasmids encoding myocardin, SRF, or myocardin and SRF. At 48 h posttransfection, the cells were harvested and RNA was prepared. Quantitative real-time PCR was performed with Applied Biosystems SYBR Green PCR Master Mix and MJ Research DNA Engine Opticon 2 real-time detection system. All RT-PCRs were performed in duplicate with (+) and without (−) RT controls. Primer pairs were designed to quantitatively amplify the mouse myocardin, SM22α, SM-α-actin, calponin-h1,

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Staining, Activity Assay, Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction

    22) Product Images from "Activation of ethylene-responsive p-hydroxyphenylpyruvate dioxygenase leads to increased tocopherol levels during ripening in mango"

    Article Title: Activation of ethylene-responsive p-hydroxyphenylpyruvate dioxygenase leads to increased tocopherol levels during ripening in mango

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/err006

    Ethylene inducibility of MiHPPD in fruit analysed by qRT-PCR. Transcript abundance of MiHPPD mRNA in mature green fruitafter; treatment with ethylene (0–24 h). Mature green fruit were exposed to ethylene for the time intervals described in the figure and RNA isolated from the samples. Expression analysis was carried out by qRT-PCR using actin as an internal control. Five fruits were used for each treatment and RNA was isolated from pooled samples. Data from three independent experiments were analysed and expressed as mean ± standard deviation. Letters (a–d) over the bars indicate significant differences at P
    Figure Legend Snippet: Ethylene inducibility of MiHPPD in fruit analysed by qRT-PCR. Transcript abundance of MiHPPD mRNA in mature green fruitafter; treatment with ethylene (0–24 h). Mature green fruit were exposed to ethylene for the time intervals described in the figure and RNA isolated from the samples. Expression analysis was carried out by qRT-PCR using actin as an internal control. Five fruits were used for each treatment and RNA was isolated from pooled samples. Data from three independent experiments were analysed and expressed as mean ± standard deviation. Letters (a–d) over the bars indicate significant differences at P

    Techniques Used: Quantitative RT-PCR, Isolation, Expressing, Standard Deviation

    23) Product Images from "Low-level shRNA Cytotoxicity Can Contribute to MYC-induced Hepatocellular Carcinoma in Adult Mice"

    Article Title: Low-level shRNA Cytotoxicity Can Contribute to MYC-induced Hepatocellular Carcinoma in Adult Mice

    Journal: Molecular Therapy

    doi: 10.1038/mt.2009.222

    Molecular analyses of AAV/shRNA-injected livers. ( a ) SYBR Green qRT-PCR analysis of p53 mRNA expression (mean ± SD), demonstrating reduction of p53 mRNA in both MYC ON and MYC OFF livers 5 and 7 days after p53 shRNA injection. Levels of p53 remained
    Figure Legend Snippet: Molecular analyses of AAV/shRNA-injected livers. ( a ) SYBR Green qRT-PCR analysis of p53 mRNA expression (mean ± SD), demonstrating reduction of p53 mRNA in both MYC ON and MYC OFF livers 5 and 7 days after p53 shRNA injection. Levels of p53 remained

    Techniques Used: shRNA, Injection, SYBR Green Assay, Quantitative RT-PCR, Expressing

    24) Product Images from "Noncoding RNA Gas5 Is a Growth Arrest and Starvation-Associated Repressor of the Glucocorticoid Receptor"

    Article Title: Noncoding RNA Gas5 Is a Growth Arrest and Starvation-Associated Repressor of the Glucocorticoid Receptor

    Journal: Science signaling

    doi: 10.1126/scisignal.2000568

    Gas5 forms hairpin structures and interacts through its 3′ portion with the GR (A) Gas5 is associated with GR through its nucleotides 400 to 598. HeLa cells were transfected with plasmids expressing the indicated fragments of Gas5 and treated with 10 −6 M of dexamethasone and RNA/protein coprecipitation assays were performed. GR-associated Gas5 fragments were detected with SYBR Green real-time PCR. The forward primer recognizing the plasmid sequence adjacent to 5′ end of the Gas5 sequence and the reverse primers in the Gas5 sequence were used to amplify exogenously expressed Gas5 fragments. ( B ) Gas5 suppresses GR-induced transcriptional activity though nucleotides 400 to 598. HeLa cells were transfected with plasmids expressing the indicated fragments of Gas5 together with the GR-expressing plasmid, pMMTV-Luc, and pSV40-β-Gal. Bars represent the mean +/− SEM values of luciferase activity normalized for β-galactosidase activity (n=3). (C) Summary of functional and physical interactions between Gas5 and the GR. Results from the coimmunoprecipitation and reporter assays both indicate that Gas5 interacts with GR through the region enclosed by its nucleotides 400 to 598. IP, immunoprecipitation. ( D) ). Wobble base-pairing is indicated in green, and Watson-Crick base-pairing is shown with blue and red. (E) Double-stranded GRE is required for Gas5 to repress GR-induced transcriptional activity. HCT116 cells were transfected with the indicated Gas5-expressing plasmid together with pRShGRα and pMMTV-Luc in the presence of pSV40-β-Gal. Luciferase activity is shown in the left panel, and RNA expression of Gas5 wild type (WT) and mutants measured with SYBR Green real-time PCR are shown in the right panel. Bars represent the mean +/− SEM values of luciferase activity normalized for β-galactosidase activity or fold Gas5 expression compared to the baseline (Gas5 WT transfection in the absence of dexamethasone). *, p
    Figure Legend Snippet: Gas5 forms hairpin structures and interacts through its 3′ portion with the GR (A) Gas5 is associated with GR through its nucleotides 400 to 598. HeLa cells were transfected with plasmids expressing the indicated fragments of Gas5 and treated with 10 −6 M of dexamethasone and RNA/protein coprecipitation assays were performed. GR-associated Gas5 fragments were detected with SYBR Green real-time PCR. The forward primer recognizing the plasmid sequence adjacent to 5′ end of the Gas5 sequence and the reverse primers in the Gas5 sequence were used to amplify exogenously expressed Gas5 fragments. ( B ) Gas5 suppresses GR-induced transcriptional activity though nucleotides 400 to 598. HeLa cells were transfected with plasmids expressing the indicated fragments of Gas5 together with the GR-expressing plasmid, pMMTV-Luc, and pSV40-β-Gal. Bars represent the mean +/− SEM values of luciferase activity normalized for β-galactosidase activity (n=3). (C) Summary of functional and physical interactions between Gas5 and the GR. Results from the coimmunoprecipitation and reporter assays both indicate that Gas5 interacts with GR through the region enclosed by its nucleotides 400 to 598. IP, immunoprecipitation. ( D) ). Wobble base-pairing is indicated in green, and Watson-Crick base-pairing is shown with blue and red. (E) Double-stranded GRE is required for Gas5 to repress GR-induced transcriptional activity. HCT116 cells were transfected with the indicated Gas5-expressing plasmid together with pRShGRα and pMMTV-Luc in the presence of pSV40-β-Gal. Luciferase activity is shown in the left panel, and RNA expression of Gas5 wild type (WT) and mutants measured with SYBR Green real-time PCR are shown in the right panel. Bars represent the mean +/− SEM values of luciferase activity normalized for β-galactosidase activity or fold Gas5 expression compared to the baseline (Gas5 WT transfection in the absence of dexamethasone). *, p

    Techniques Used: Transfection, Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Activity Assay, Luciferase, Functional Assay, Immunoprecipitation, RNA Expression

    Gas5 binds GR DBD through its double-stranded RNA GRE-mimic located at hairpin #5 and competes with DNA GREs for binding to GR DBD in vitro ( A, B ) Gas5 directly binds GR DBD through its double-stranded RNA GRE-mimic in an in vitro binding assay. 10 −4 M of indicated Gas5 RNAs was incubated with 1 μg of GST or GST-fused GR DBD bound to GST beads in the presence or absence of increasing concentrations (0, 10 −5 , 10 −4 , 10 −3 , and 10 −2 ) of the double-stranded DNA GREs. Associated RNA was measured with the SYBR Green real-time PCR. Bars and circles represent the mean +/− SEM values of fold change in Gas5 association compared to the baseline (GST) and of the Gas5 molecules associated with GR DBD, respectively. ( B) is a Scatchard plot (n=3). (C) Gas5 competes with FAM-labeled DNA GREs for binding to the GR DBD through its RNA GRE-mimic in vitro. Oneμg of GST-fused GR DBD bound to the GST beads was incubated with 10 −7 M of FAM-labeled double-stranded GREs in the presence of the double-stranded GREs, wild type (WT) Gas5 or Gas5 GRE-1 Mut RNAs. Symbols represent the mean +/− SEM values of % binding obtained by dividing the FAM-oriented fluorescence intensity of each point with those of the baseline (in the absence of competitors) (n=3). (D) ). The analogous base pairs in the Gas5 GRE-mimic (G540 and C554, respectively, shown in red) are calculated to be oriented similarly to those in the native DNA, possibly enabling them to form the hydrogen bonds with K442 and R447 of the GR DBD, respectively. The analogous base pairs in the Gas5 RNA MRE-resembling region (U453 and G474, shown in red) are calculated to be poorly oriented for potential binding to GR DBD. ( F: Replacement of K442, R447, or both of GR DBD attenuates interaction of GST-GR DBD to Gas5 in vitro. 10 −4 M of wild type (WT) Gas5 RNA was incubated with 1 μg of GST or the indicated GST-fused GR DBDs bound to GST beads. Associated Gas5 RNA was measured with SYBR Green real-time PCR. Bars represent the mean +/− SEM values of fold change in the association of Gas5 RNA normalized to baseline (with GST). *, p
    Figure Legend Snippet: Gas5 binds GR DBD through its double-stranded RNA GRE-mimic located at hairpin #5 and competes with DNA GREs for binding to GR DBD in vitro ( A, B ) Gas5 directly binds GR DBD through its double-stranded RNA GRE-mimic in an in vitro binding assay. 10 −4 M of indicated Gas5 RNAs was incubated with 1 μg of GST or GST-fused GR DBD bound to GST beads in the presence or absence of increasing concentrations (0, 10 −5 , 10 −4 , 10 −3 , and 10 −2 ) of the double-stranded DNA GREs. Associated RNA was measured with the SYBR Green real-time PCR. Bars and circles represent the mean +/− SEM values of fold change in Gas5 association compared to the baseline (GST) and of the Gas5 molecules associated with GR DBD, respectively. ( B) is a Scatchard plot (n=3). (C) Gas5 competes with FAM-labeled DNA GREs for binding to the GR DBD through its RNA GRE-mimic in vitro. Oneμg of GST-fused GR DBD bound to the GST beads was incubated with 10 −7 M of FAM-labeled double-stranded GREs in the presence of the double-stranded GREs, wild type (WT) Gas5 or Gas5 GRE-1 Mut RNAs. Symbols represent the mean +/− SEM values of % binding obtained by dividing the FAM-oriented fluorescence intensity of each point with those of the baseline (in the absence of competitors) (n=3). (D) ). The analogous base pairs in the Gas5 GRE-mimic (G540 and C554, respectively, shown in red) are calculated to be oriented similarly to those in the native DNA, possibly enabling them to form the hydrogen bonds with K442 and R447 of the GR DBD, respectively. The analogous base pairs in the Gas5 RNA MRE-resembling region (U453 and G474, shown in red) are calculated to be poorly oriented for potential binding to GR DBD. ( F: Replacement of K442, R447, or both of GR DBD attenuates interaction of GST-GR DBD to Gas5 in vitro. 10 −4 M of wild type (WT) Gas5 RNA was incubated with 1 μg of GST or the indicated GST-fused GR DBDs bound to GST beads. Associated Gas5 RNA was measured with SYBR Green real-time PCR. Bars represent the mean +/− SEM values of fold change in the association of Gas5 RNA normalized to baseline (with GST). *, p

    Techniques Used: Binding Assay, In Vitro, Incubation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Labeling, Fluorescence

    Endogenous Gas5 functions as a negative regulator of GR-induced cIAP2 and SGK1 mRNA expression HeLa cells were transfected with Gas5 or control siRNA and were cultured in serum-free medium for the indicated time periods. The abundance of the cIAP2 , SGK1 , and RPLP0 mRNAs and of the Gas5 RNA were determined with SYBR Green real-time PCR. ( A ) Thymidine incorporation was also examined in cells from the same experiment and is shown as % of the baseline. Bars represent the mean +/− SEM values of fold induction of the Gas5 RNA ( B ) and the cIAP2 ( C ) and SGK1 ( D ) mRNAs normalized for RPLP0 mRNA abundance compared to the baseline (in the absence of Gas5 siRNA and dexamethasone at time “0”). (n=3).
    Figure Legend Snippet: Endogenous Gas5 functions as a negative regulator of GR-induced cIAP2 and SGK1 mRNA expression HeLa cells were transfected with Gas5 or control siRNA and were cultured in serum-free medium for the indicated time periods. The abundance of the cIAP2 , SGK1 , and RPLP0 mRNAs and of the Gas5 RNA were determined with SYBR Green real-time PCR. ( A ) Thymidine incorporation was also examined in cells from the same experiment and is shown as % of the baseline. Bars represent the mean +/− SEM values of fold induction of the Gas5 RNA ( B ) and the cIAP2 ( C ) and SGK1 ( D ) mRNAs normalized for RPLP0 mRNA abundance compared to the baseline (in the absence of Gas5 siRNA and dexamethasone at time “0”). (n=3).

    Techniques Used: Expressing, Transfection, Cell Culture, SYBR Green Assay, Real-time Polymerase Chain Reaction

    Gas5 RNA interacts with GR at its DBD and suppresses its transcriptional activity (A) Endogenous Gas5 interacts with GR in a dexamethasone-dependent fashion. GR-expressing HeLa cells were treated with 10 −6 M of dexamethasone. The GR/RNA complex was precipitated with a specific antibody against GR LBD (anti-GR Ab), and rabbit IgG was used as a negative control. GR-associated Gas5 was detected with regular PCR (left panel) employing an adjusted PCR cycle and a primer pair that amplifies the entire Gas5 sequence or with SYBR Green real-time PCR (middle panel) using a specific primer pair. Results of Western blots show the abundance of the GR protein (bottom gel of the left panel). The right panel shows titration curves of dexamethasone for the association of Gas5 to GR examined with anti-GR (closed circles) or control (open circles) antibody. Bars and circles represent the mean +/− SEM values of the fold Gas5 precipitation over the baseline (“Control” in the absence of dexamethasone) (n=3). IP Ab: antibody used for immunoprecipitation. (B, C) GR is associated with Gas5 and t RNA(Arg) in a ligand-dependent fashion, whereas control RNAs miR191, SC35 mRNA, and U75 snoRNA are not. HeLa cells were treated with 10 −6 M of dexamethasone. GR-associated Gas5, miR191, and SC35 mRNA ( B ), or U75 snoRNA and t RNA(Arg) mRNA ( C ) were detected with regular PCR employing an adjusted PCR cycle and specific primer pairs that amplify the entire sequences. Results of Western blots demonstrating GR protein abundance are shown in the bottom gels. (D) Gas5 is associated with the GR DBD. GR-deficient HCT116 cells were transfected with the control plasmid (Vector), pRShGRα (GR WT), or G-gal-G, and treated with 10 −6 M of dexamethasone. Gas5 coprecipitated with GR or G-gal-G with a GR LBD-specific antibody (anti-GR Ab) was detected with regular PCR (left panel) employing an adjusted PCR cycle and a primer pair that amplifies the entire Gas5 sequence or with SYBR Green real-time PCR (right panel) using a specific primer pair. Western blots showing the abundance of GR WT and G-gal-G are provided at the bottom of the left panel. Bars represent the mean +/− SEM values of fold Gas5 precipitation over the baseline (“Vector” in the absence of dexamethasone) (n=3). IP: immunoprecipitation. (E) Gas5 suppresses the transcriptional activity of wild type (WT) GR but not of the GR chimera containing the GAL4 DBD. HCT116 cells were transfected with the Gas5-expressing plasmid together with pRShGRα and pMMTV-Luc, or G-gal-G and p17mer-tk-Luc, in the presence of pSV40-β-Gal. Western blots showing GR WT and G-gal-G abundance are provided at the bottom. Bars represent the mean +/− SEM values of luciferase activity normalized to β-galactosidase activity. *; p
    Figure Legend Snippet: Gas5 RNA interacts with GR at its DBD and suppresses its transcriptional activity (A) Endogenous Gas5 interacts with GR in a dexamethasone-dependent fashion. GR-expressing HeLa cells were treated with 10 −6 M of dexamethasone. The GR/RNA complex was precipitated with a specific antibody against GR LBD (anti-GR Ab), and rabbit IgG was used as a negative control. GR-associated Gas5 was detected with regular PCR (left panel) employing an adjusted PCR cycle and a primer pair that amplifies the entire Gas5 sequence or with SYBR Green real-time PCR (middle panel) using a specific primer pair. Results of Western blots show the abundance of the GR protein (bottom gel of the left panel). The right panel shows titration curves of dexamethasone for the association of Gas5 to GR examined with anti-GR (closed circles) or control (open circles) antibody. Bars and circles represent the mean +/− SEM values of the fold Gas5 precipitation over the baseline (“Control” in the absence of dexamethasone) (n=3). IP Ab: antibody used for immunoprecipitation. (B, C) GR is associated with Gas5 and t RNA(Arg) in a ligand-dependent fashion, whereas control RNAs miR191, SC35 mRNA, and U75 snoRNA are not. HeLa cells were treated with 10 −6 M of dexamethasone. GR-associated Gas5, miR191, and SC35 mRNA ( B ), or U75 snoRNA and t RNA(Arg) mRNA ( C ) were detected with regular PCR employing an adjusted PCR cycle and specific primer pairs that amplify the entire sequences. Results of Western blots demonstrating GR protein abundance are shown in the bottom gels. (D) Gas5 is associated with the GR DBD. GR-deficient HCT116 cells were transfected with the control plasmid (Vector), pRShGRα (GR WT), or G-gal-G, and treated with 10 −6 M of dexamethasone. Gas5 coprecipitated with GR or G-gal-G with a GR LBD-specific antibody (anti-GR Ab) was detected with regular PCR (left panel) employing an adjusted PCR cycle and a primer pair that amplifies the entire Gas5 sequence or with SYBR Green real-time PCR (right panel) using a specific primer pair. Western blots showing the abundance of GR WT and G-gal-G are provided at the bottom of the left panel. Bars represent the mean +/− SEM values of fold Gas5 precipitation over the baseline (“Vector” in the absence of dexamethasone) (n=3). IP: immunoprecipitation. (E) Gas5 suppresses the transcriptional activity of wild type (WT) GR but not of the GR chimera containing the GAL4 DBD. HCT116 cells were transfected with the Gas5-expressing plasmid together with pRShGRα and pMMTV-Luc, or G-gal-G and p17mer-tk-Luc, in the presence of pSV40-β-Gal. Western blots showing GR WT and G-gal-G abundance are provided at the bottom. Bars represent the mean +/− SEM values of luciferase activity normalized to β-galactosidase activity. *; p

    Techniques Used: Activity Assay, Expressing, Negative Control, Polymerase Chain Reaction, Sequencing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Western Blot, Titration, Immunoprecipitation, Transfection, Plasmid Preparation, Luciferase

    Gas5 is located both in the cytoplasm and the nucleus and translocates from the cytoplasm into the nucleus with GR in response to dexamethasone (A) Gas5 is localized both in the cytoplasm and the nucleus. Top left shows RNA fluorescence in situ hybridization performed with the Alexa488-labeled Gas5 probe in HeLa cells. Bottom left shows the negative control obtained with samples treated with RNase. Middle panels demonstrate staining with DAPI, and the right panels show merged images of left and middle panels. (B) Gas5 translocates into the nucleus with GR in response to dexamethasone. HeLa cells were transfected with Gas5 wild type (WT)- or GRE-1 Mut-expressing plasmid and were treated with 10 −6 M of dexamethasone. Transfected Gas5s and endogenous GR, α-tubulin, and Oct1 in these subcellular fractions were detected with SYBR Green real-time PCR and Western blots, respectively (top panels). The bottom panel shows Western blots for the presence of α-tubulin and Oct1 in the subcellular fractions. To detect exogenously expressed Gas5, we employed the forward primer, which recognized the plasmid sequence adjacent to 5′ end of the Gas5 sequence that was expressed with Gas5 RNA. Bars represent the mean +/− SEM values of fold accumulation of Gas5 RNA compared to the baseline (transfected with wild type Gas5 RNA in the absence of dexamethasone). *: p
    Figure Legend Snippet: Gas5 is located both in the cytoplasm and the nucleus and translocates from the cytoplasm into the nucleus with GR in response to dexamethasone (A) Gas5 is localized both in the cytoplasm and the nucleus. Top left shows RNA fluorescence in situ hybridization performed with the Alexa488-labeled Gas5 probe in HeLa cells. Bottom left shows the negative control obtained with samples treated with RNase. Middle panels demonstrate staining with DAPI, and the right panels show merged images of left and middle panels. (B) Gas5 translocates into the nucleus with GR in response to dexamethasone. HeLa cells were transfected with Gas5 wild type (WT)- or GRE-1 Mut-expressing plasmid and were treated with 10 −6 M of dexamethasone. Transfected Gas5s and endogenous GR, α-tubulin, and Oct1 in these subcellular fractions were detected with SYBR Green real-time PCR and Western blots, respectively (top panels). The bottom panel shows Western blots for the presence of α-tubulin and Oct1 in the subcellular fractions. To detect exogenously expressed Gas5, we employed the forward primer, which recognized the plasmid sequence adjacent to 5′ end of the Gas5 sequence that was expressed with Gas5 RNA. Bars represent the mean +/− SEM values of fold accumulation of Gas5 RNA compared to the baseline (transfected with wild type Gas5 RNA in the absence of dexamethasone). *: p

    Techniques Used: Fluorescence, In Situ Hybridization, Labeling, Negative Control, Staining, Transfection, Expressing, Plasmid Preparation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Western Blot, Sequencing

    Overexpression of Gas5 suppresses both the association of GR with cIAP2 GREs and GR-induced cIAP2 mRNA expression HeLa cells were transfected with increasing amounts of Gas5-expressing plasmid and treated with 10 −6 M of dexamethasone. The ChIP assay and total RNA purification were performed using fractions of these cells. (A) Gas5 suppresses the association of GR with cIAP2 GREs. ChIP assays were performed with the GR antibody against GR LBD (anti-GR Ab) and the cIAP2 promoter fragment that contains tandem GREs was amplified by PCR with an adjusted PCR cycle using a specific primer pair. The bottom blots are Western blots showing GR protein abundance. (B and C) Gas5 suppresses dexamethasone-induced cIAP2 mRNA expression. Total RNA was purified, and the abundance of the cIAP2 and control RPLP0 mRNAs and of the Gas5 RNA were measured with SYBR Green real-time PCR. Bars represent the mean +/− SEM values of fold induction of the cIAP2 mRNA (B) and the Gas5 RNA (C) expression normalized for the RPLP0 mRNA abundance compared to the baseline (in the absence of Gas5 transfection and dexamethasone). *, p
    Figure Legend Snippet: Overexpression of Gas5 suppresses both the association of GR with cIAP2 GREs and GR-induced cIAP2 mRNA expression HeLa cells were transfected with increasing amounts of Gas5-expressing plasmid and treated with 10 −6 M of dexamethasone. The ChIP assay and total RNA purification were performed using fractions of these cells. (A) Gas5 suppresses the association of GR with cIAP2 GREs. ChIP assays were performed with the GR antibody against GR LBD (anti-GR Ab) and the cIAP2 promoter fragment that contains tandem GREs was amplified by PCR with an adjusted PCR cycle using a specific primer pair. The bottom blots are Western blots showing GR protein abundance. (B and C) Gas5 suppresses dexamethasone-induced cIAP2 mRNA expression. Total RNA was purified, and the abundance of the cIAP2 and control RPLP0 mRNAs and of the Gas5 RNA were measured with SYBR Green real-time PCR. Bars represent the mean +/− SEM values of fold induction of the cIAP2 mRNA (B) and the Gas5 RNA (C) expression normalized for the RPLP0 mRNA abundance compared to the baseline (in the absence of Gas5 transfection and dexamethasone). *, p

    Techniques Used: Over Expression, Expressing, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Purification, Amplification, Polymerase Chain Reaction, Western Blot, SYBR Green Assay, Real-time Polymerase Chain Reaction

    25) Product Images from "Differential heat shock gene hsp70-1 response to toxicants revealed by in vivo study of lungs in transgenic mice"

    Article Title: Differential heat shock gene hsp70-1 response to toxicants revealed by in vivo study of lungs in transgenic mice

    Journal: Cell Stress & Chaperones

    doi:

    Kinetics of HSP70.1 induction by heat shock (48°C for 15 minute). (A) Analysis of endogenous hsp70-1 mRNA was evaluated by SYBR Green Real-Time Quantitative RT-PCR. Level of mRNA expression is expressed as arbitrary units (AU) corresponding to the ratio (RT-PCR signal for the Hsp70.1 mRNA)/(RT-PCR signal for the 18S rRNA). Two animals were analyzed for each time period tested in duplicate. Values represent the means ± SE. (B) Analysis of HSP70.1Luc expression in lung was assessed by measurement of luciferase activity (RLU/μg of protein) in tissue extracts. Each value represents the means ± SE calculated from measurements made with n = 5 mice for each time period tested. (*, P
    Figure Legend Snippet: Kinetics of HSP70.1 induction by heat shock (48°C for 15 minute). (A) Analysis of endogenous hsp70-1 mRNA was evaluated by SYBR Green Real-Time Quantitative RT-PCR. Level of mRNA expression is expressed as arbitrary units (AU) corresponding to the ratio (RT-PCR signal for the Hsp70.1 mRNA)/(RT-PCR signal for the 18S rRNA). Two animals were analyzed for each time period tested in duplicate. Values represent the means ± SE. (B) Analysis of HSP70.1Luc expression in lung was assessed by measurement of luciferase activity (RLU/μg of protein) in tissue extracts. Each value represents the means ± SE calculated from measurements made with n = 5 mice for each time period tested. (*, P

    Techniques Used: SYBR Green Assay, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Mouse Assay

    Quantitative analysis of hsp70-1 gene response to toxicant exposure. hsp70-1 transcripts in mouse lungs were analyzed by SYBR Green Real-Time Quantitative RT-PCR after HS, heat shock (48°C, 15 minute); Cd, cadmium chloride (5 mg/kg); O 3 , ozone (1 ppm); PQ, paraquat (20 mg/kg), or Pth, parathion (1 mg/kg) exposure. Lungs were harvested at 1 hour, 3 hours, or 5 hours after toxicant exposure. Data represent mRNA level expressed as arbitrary units (AU) corresponding to the ratio (RT-PCR signal for the Hsp70.1 mRNA)/(RT-PCR signal for the 18S rRNA). Two animals were analyzed for each time period tested in duplicate. Values represent the means ± standard error. (*, P
    Figure Legend Snippet: Quantitative analysis of hsp70-1 gene response to toxicant exposure. hsp70-1 transcripts in mouse lungs were analyzed by SYBR Green Real-Time Quantitative RT-PCR after HS, heat shock (48°C, 15 minute); Cd, cadmium chloride (5 mg/kg); O 3 , ozone (1 ppm); PQ, paraquat (20 mg/kg), or Pth, parathion (1 mg/kg) exposure. Lungs were harvested at 1 hour, 3 hours, or 5 hours after toxicant exposure. Data represent mRNA level expressed as arbitrary units (AU) corresponding to the ratio (RT-PCR signal for the Hsp70.1 mRNA)/(RT-PCR signal for the 18S rRNA). Two animals were analyzed for each time period tested in duplicate. Values represent the means ± standard error. (*, P

    Techniques Used: SYBR Green Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    26) Product Images from "AMPK Regulates Metabolic Actions of Glucocorticoids by Phosphorylating the Glucocorticoid Receptor through p38 MAPK"

    Article Title: AMPK Regulates Metabolic Actions of Glucocorticoids by Phosphorylating the Glucocorticoid Receptor through p38 MAPK

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2010-0192

    SYBR Green-based real-time PCR
    Figure Legend Snippet: SYBR Green-based real-time PCR

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction

    27) Product Images from "The Arabidopsis Resistance-Like Gene SNC1 Is Activated by Mutations in SRFR1 and Contributes to Resistance to the Bacterial Effector AvrRps4SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity"

    Article Title: The Arabidopsis Resistance-Like Gene SNC1 Is Activated by Mutations in SRFR1 and Contributes to Resistance to the Bacterial Effector AvrRps4SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001172

    SNC1-RLD encodes a truncated TNL protein. (A) Alignment of deduced amino acid sequences of SNC1-Col (top) and SNC1-RLD (bottom) using the EBI-ClustalW tool ( http://www.ebi.ac.uk/Tools/clustalw/ ) [54] . Identical amino acids are indicated by asterisks. Colons and semi-colons show conserved substitutions and semi-conserved substitutions, respectively. Characters in blue, red and green show the amino acids corresponding to exon 1, exon 2 and exon 3, respectively. (B) SNC1 gene model as experimentally verified by reverse transcription PCR and 3′-RACE from Col-0 (middle) and RLD (bottom) compared with the TAIR9 gene model (top). Exons are indicated by boxes, introns by lines, and stop codons by red asterisks.
    Figure Legend Snippet: SNC1-RLD encodes a truncated TNL protein. (A) Alignment of deduced amino acid sequences of SNC1-Col (top) and SNC1-RLD (bottom) using the EBI-ClustalW tool ( http://www.ebi.ac.uk/Tools/clustalw/ ) [54] . Identical amino acids are indicated by asterisks. Colons and semi-colons show conserved substitutions and semi-conserved substitutions, respectively. Characters in blue, red and green show the amino acids corresponding to exon 1, exon 2 and exon 3, respectively. (B) SNC1 gene model as experimentally verified by reverse transcription PCR and 3′-RACE from Col-0 (middle) and RLD (bottom) compared with the TAIR9 gene model (top). Exons are indicated by boxes, introns by lines, and stop codons by red asterisks.

    Techniques Used: Polymerase Chain Reaction

    28) Product Images from "CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use"

    Article Title: CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-6-5

    Comparison to TaqMan ® technology. Total RNA was extracted from isolated monocytes stimulated for 6 hours by LPS, serial-diluted 1:10 and retro-transcribed to generate standard curves by plotting the C T against the concentration of this cellular RNA in arbitrary units. TNF-α transcripts were amplified by real time PCR using either ( A ) the TaqMan ® commercial kit and the TaqMan ® universal PCR master mix or ( B ) our primers and the SYBR Green PCR master mix. Data represent the standard curves obtained for the two techniques, their slopes and the deducted efficiencies.
    Figure Legend Snippet: Comparison to TaqMan ® technology. Total RNA was extracted from isolated monocytes stimulated for 6 hours by LPS, serial-diluted 1:10 and retro-transcribed to generate standard curves by plotting the C T against the concentration of this cellular RNA in arbitrary units. TNF-α transcripts were amplified by real time PCR using either ( A ) the TaqMan ® commercial kit and the TaqMan ® universal PCR master mix or ( B ) our primers and the SYBR Green PCR master mix. Data represent the standard curves obtained for the two techniques, their slopes and the deducted efficiencies.

    Techniques Used: Isolation, Concentration Assay, Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, SYBR Green Assay

    Primer validation. A. Agarose gel electrophoresis of several CyProQuant-PCR products generated by amplification of a pool of cDNA from PBMC stimulated in vitro by LPS or PHA. Scale is shown in base pairs (bp). B. Dissociation curve analysis of these CyProQuant-PCR products. Negative derivative of the fluorescence is plotted against temperature. The single peak shows that SYBR Green fluorescence detects only the specific CyProQuant-PCR product. C. Amplification plots and standard curve resulting from the amplification of a range of β2-MG external RNA standard using CyProQuant-PCR.
    Figure Legend Snippet: Primer validation. A. Agarose gel electrophoresis of several CyProQuant-PCR products generated by amplification of a pool of cDNA from PBMC stimulated in vitro by LPS or PHA. Scale is shown in base pairs (bp). B. Dissociation curve analysis of these CyProQuant-PCR products. Negative derivative of the fluorescence is plotted against temperature. The single peak shows that SYBR Green fluorescence detects only the specific CyProQuant-PCR product. C. Amplification plots and standard curve resulting from the amplification of a range of β2-MG external RNA standard using CyProQuant-PCR.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Generated, Amplification, In Vitro, Fluorescence, SYBR Green Assay

    29) Product Images from "Myoblast-Derived Neuronal Cells Form Glutamatergic Neurons in the Mouse Cerebellum"

    Article Title: Myoblast-Derived Neuronal Cells Form Glutamatergic Neurons in the Mouse Cerebellum

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.509

    M-RV-GFP(T) cells exhibit synaptic communication and express neuronal differentiation markers similar to native granule neurons. ( A ): An action potential in representative green fluorescent protein (GFP)-positive cells identified using an Olympus VX50WI fluorescence microscope. ( B ): Miniature excitatory postsynaptic currents (EPSCs) in representative GFP-positive M-RV-GFP(T) cells proximal to the injection tract and native GFP-negative CGNs distal to the injection tract. EPSCs in GFP-positive cells and CGNs were abolished by the NMDA and nonNMDA receptor antagonists 2-amino-5-phosphovalerate (10 μ M) and 6-cyano-7-nitroquinoxaline-2,3-dione (10 μ M), respectively, in the bath solution. ( C ): Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was carried out to amplify REST-VP16 transgene sequences using RNA prepared from M-RV-GFP(T) single cells in which EPSC was measured. In vitro-cultured differentiated M-RV-GFP (M-RV-GFP[D]) cells were used as the positive control for the detection of REST-VP16 sequences and the neuronal differentiation marker SCG10. Endogenous CGNs were used as the negative control for REST-VP16 expression and positive control for SCG10 expression. GAPDH served as the internal control. One-fifth of the PCR product was loaded for M-RV-GFP(D) cells, and one-half of the reactions using M-RV-GFP(T) cells and CGNs were loaded on 2.5% agarose gels for electrophoresis. ( D ): Quantitative SYBR green RT-PCR was used to measure the expression of REST-VP16 , GLU-R2 , GAD1 , and SCG10 in single cell preparations, as described in ( B ). GAPDH was used as an internal control and to normalize gene expression. All reactions were carried out in triplicate, and standard deviation calculated. Abbreviations: GAD1, glutamate decarboxylase 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GCN, granule cell neuron; Glu2, glutamate receptor 2; M-RV-GFP(T), transplanted green fluorescent protein (GFP)-tagged M-RV cells.
    Figure Legend Snippet: M-RV-GFP(T) cells exhibit synaptic communication and express neuronal differentiation markers similar to native granule neurons. ( A ): An action potential in representative green fluorescent protein (GFP)-positive cells identified using an Olympus VX50WI fluorescence microscope. ( B ): Miniature excitatory postsynaptic currents (EPSCs) in representative GFP-positive M-RV-GFP(T) cells proximal to the injection tract and native GFP-negative CGNs distal to the injection tract. EPSCs in GFP-positive cells and CGNs were abolished by the NMDA and nonNMDA receptor antagonists 2-amino-5-phosphovalerate (10 μ M) and 6-cyano-7-nitroquinoxaline-2,3-dione (10 μ M), respectively, in the bath solution. ( C ): Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was carried out to amplify REST-VP16 transgene sequences using RNA prepared from M-RV-GFP(T) single cells in which EPSC was measured. In vitro-cultured differentiated M-RV-GFP (M-RV-GFP[D]) cells were used as the positive control for the detection of REST-VP16 sequences and the neuronal differentiation marker SCG10. Endogenous CGNs were used as the negative control for REST-VP16 expression and positive control for SCG10 expression. GAPDH served as the internal control. One-fifth of the PCR product was loaded for M-RV-GFP(D) cells, and one-half of the reactions using M-RV-GFP(T) cells and CGNs were loaded on 2.5% agarose gels for electrophoresis. ( D ): Quantitative SYBR green RT-PCR was used to measure the expression of REST-VP16 , GLU-R2 , GAD1 , and SCG10 in single cell preparations, as described in ( B ). GAPDH was used as an internal control and to normalize gene expression. All reactions were carried out in triplicate, and standard deviation calculated. Abbreviations: GAD1, glutamate decarboxylase 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GCN, granule cell neuron; Glu2, glutamate receptor 2; M-RV-GFP(T), transplanted green fluorescent protein (GFP)-tagged M-RV cells.

    Techniques Used: Fluorescence, Microscopy, Injection, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, In Vitro, Cell Culture, Positive Control, Marker, Negative Control, Expressing, Electrophoresis, SYBR Green Assay, Standard Deviation

    30) Product Images from "The PTH-Gαs-Protein Kinase A Cascade Controls αNAC Localization To Regulate Bone Mass"

    Article Title: The PTH-Gαs-Protein Kinase A Cascade Controls αNAC Localization To Regulate Bone Mass

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01434-13

    PTH-mediated αNAC accumulation at the Ocn promoter through a PKA-dependent pathway. (A to C) Quantitative chromatin immunoprecipitation (ChIP) assays in cells treated with vehicle, 100 nM PTH(1–34), or 100 μM 6Bnz-cAMP for 30 min. (A) MC3T3-E1 cells; (B) UMR-106 cells stably infected with an empty vector or a dominant-negative form of PKA (PKAdn); (C) UMR-106 cells stably expressing FLAG-tagged wild-type (WT) or S99A mutant αNAC. Quantitative ChIP assays were performed with anti-αNAC or anti-FLAG epitope antibody. ChIP products were amplified by SYBR green PCR using specific primers flanking the αNAC binding site in the Ocn promoter. Relative promoter occupancy was calculated as enrichment over vehicle-treated cells, which was ascribed an arbitrary value of 1. Results are means ± SEM of triplicate determinations. Neg. Ctrl, negative-control ChIP performed with naive IgG on cells treated for 30 min with 6Bnz-cAMP. (D) Reporter gene transient-transfection assay. UMR-106 cells were transfected with an Ocn -luc reporter plasmid and expression vectors for c-JUN in combination with wild-type αNAC or S99A or S99D mutant αNAC. Results are mean fold induction ± SEM, with luciferase levels measured in cells transfected with the empty expression vectors arbitrarily ascribed a value of 1. *, P
    Figure Legend Snippet: PTH-mediated αNAC accumulation at the Ocn promoter through a PKA-dependent pathway. (A to C) Quantitative chromatin immunoprecipitation (ChIP) assays in cells treated with vehicle, 100 nM PTH(1–34), or 100 μM 6Bnz-cAMP for 30 min. (A) MC3T3-E1 cells; (B) UMR-106 cells stably infected with an empty vector or a dominant-negative form of PKA (PKAdn); (C) UMR-106 cells stably expressing FLAG-tagged wild-type (WT) or S99A mutant αNAC. Quantitative ChIP assays were performed with anti-αNAC or anti-FLAG epitope antibody. ChIP products were amplified by SYBR green PCR using specific primers flanking the αNAC binding site in the Ocn promoter. Relative promoter occupancy was calculated as enrichment over vehicle-treated cells, which was ascribed an arbitrary value of 1. Results are means ± SEM of triplicate determinations. Neg. Ctrl, negative-control ChIP performed with naive IgG on cells treated for 30 min with 6Bnz-cAMP. (D) Reporter gene transient-transfection assay. UMR-106 cells were transfected with an Ocn -luc reporter plasmid and expression vectors for c-JUN in combination with wild-type αNAC or S99A or S99D mutant αNAC. Results are mean fold induction ± SEM, with luciferase levels measured in cells transfected with the empty expression vectors arbitrarily ascribed a value of 1. *, P

    Techniques Used: Chromatin Immunoprecipitation, Stable Transfection, Infection, Plasmid Preparation, Dominant Negative Mutation, Expressing, Mutagenesis, FLAG-tag, Amplification, SYBR Green Assay, Polymerase Chain Reaction, Binding Assay, Negative Control, Transient Transfection Assay, Transfection, Luciferase

    31) Product Images from "Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA"

    Article Title: Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000965

    Inhibition of deacetylase activity fails to rescue the replication of pUL29/28 or pUL38-deficient viruses. (A) Trichostatin A does not rescue pUL29/28 or pUL38-deficient viruses. Fibroblasts were infected at a multiplicity of 0.1 pfu/cell using wild-type (BAD wt ) or inoculum containing equivalent numbers of viral genomes for BAD sub UL29 ( sub UL29) or BAD sub UL38 ( sub UL38) viruses. Cultures were treated with 300 nM TSA for the duration of the experiment and viral titers from culture supernatants were determined at 10 dpi. Data is from duplicate experiments. (B) BAD sub UL29 ( sub UL29) virus is deficient in immediate-early gene expression irrespective of treatment with TSA. Cells pretreated with 500 nM of TSA were infected using 0.1 pfu/cell of wild-type (BAD w t) or BAD sub UL29 ( sub UL29) virus as described above, and total RNA was harvested at the indicated times. Expression of IE1 RNA was determined by qRT-PCR using cDNA produced from DNase-treated RNA and random hexamers. Quantitation was completed with SYBR green and primers specific to exon 4 of IE1. Samples were normalized to cellular GAPDH RNA levels.
    Figure Legend Snippet: Inhibition of deacetylase activity fails to rescue the replication of pUL29/28 or pUL38-deficient viruses. (A) Trichostatin A does not rescue pUL29/28 or pUL38-deficient viruses. Fibroblasts were infected at a multiplicity of 0.1 pfu/cell using wild-type (BAD wt ) or inoculum containing equivalent numbers of viral genomes for BAD sub UL29 ( sub UL29) or BAD sub UL38 ( sub UL38) viruses. Cultures were treated with 300 nM TSA for the duration of the experiment and viral titers from culture supernatants were determined at 10 dpi. Data is from duplicate experiments. (B) BAD sub UL29 ( sub UL29) virus is deficient in immediate-early gene expression irrespective of treatment with TSA. Cells pretreated with 500 nM of TSA were infected using 0.1 pfu/cell of wild-type (BAD w t) or BAD sub UL29 ( sub UL29) virus as described above, and total RNA was harvested at the indicated times. Expression of IE1 RNA was determined by qRT-PCR using cDNA produced from DNase-treated RNA and random hexamers. Quantitation was completed with SYBR green and primers specific to exon 4 of IE1. Samples were normalized to cellular GAPDH RNA levels.

    Techniques Used: Inhibition, Histone Deacetylase Assay, Activity Assay, Infection, Expressing, Quantitative RT-PCR, Produced, Quantitation Assay, SYBR Green Assay

    Reduced expression of components of the NuRD complex inhibits HCMV replication. (A) Disruption of NuRD complex in primary fibroblasts. Short hairpin RNA (shRNA) sequences to a scrambled control, CHD4, RBBP4 or both CHD4 and RBBP4 were delivered to fibroblasts using lentivirus vectors and expressing cells were isolated by puromycin resistance. Expression of CHD4 and RBBP4 was quantified by qRT-PCR using two separate sets of gene-specific primers and total cellular RNA. The data was normalized to GAPDH RNA levels and includes the average percent reduction for both primer sets to each gene. (B) shRNA-expressing cells were infected at a multiplicity of 0.25 pfu/cell with a BAD wt derivative expressing GFP. Images of infected cells were captured at 96 hpi. (C) Expression of immediate-early and early RNAs at 10 hpi was determined by qRT-PCR using cDNA produced from DNase-treated RNA and random hexamers. Quantitation was completed with SYBR green and sequence-specific primers as indicated. Expression was normalized to cellular GAPDH RNA levels. (D) HCMV genome replication was quantified by qPCR at 96 hpi. qPCR data was normalized using primers to β-actin. Data is derived from replicate experiments.
    Figure Legend Snippet: Reduced expression of components of the NuRD complex inhibits HCMV replication. (A) Disruption of NuRD complex in primary fibroblasts. Short hairpin RNA (shRNA) sequences to a scrambled control, CHD4, RBBP4 or both CHD4 and RBBP4 were delivered to fibroblasts using lentivirus vectors and expressing cells were isolated by puromycin resistance. Expression of CHD4 and RBBP4 was quantified by qRT-PCR using two separate sets of gene-specific primers and total cellular RNA. The data was normalized to GAPDH RNA levels and includes the average percent reduction for both primer sets to each gene. (B) shRNA-expressing cells were infected at a multiplicity of 0.25 pfu/cell with a BAD wt derivative expressing GFP. Images of infected cells were captured at 96 hpi. (C) Expression of immediate-early and early RNAs at 10 hpi was determined by qRT-PCR using cDNA produced from DNase-treated RNA and random hexamers. Quantitation was completed with SYBR green and sequence-specific primers as indicated. Expression was normalized to cellular GAPDH RNA levels. (D) HCMV genome replication was quantified by qPCR at 96 hpi. qPCR data was normalized using primers to β-actin. Data is derived from replicate experiments.

    Techniques Used: Expressing, shRNA, Isolation, Quantitative RT-PCR, Infection, Produced, Quantitation Assay, SYBR Green Assay, Sequencing, Real-time Polymerase Chain Reaction, Derivative Assay

    32) Product Images from "Development and Application of a Universal Hemoplasma Screening Assay Based on the SYBR Green PCR Principle ▿"

    Article Title: Development and Application of a Universal Hemoplasma Screening Assay Based on the SYBR Green PCR Principle ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01478-09

    Melting curve analysis of SYBR green PCR products.
    Figure Legend Snippet: Melting curve analysis of SYBR green PCR products.

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction

    SYBR green PCR assay optimization.
    Figure Legend Snippet: SYBR green PCR assay optimization.

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction

    Melting curve analysis after hemoplasma SYBR green PCR of samples from animals infected singly with a feline, canine, murine, or porcine hemoplasma species. Each curve represents one PCR amplicon and depicts the change in fluorescence during a continuous
    Figure Legend Snippet: Melting curve analysis after hemoplasma SYBR green PCR of samples from animals infected singly with a feline, canine, murine, or porcine hemoplasma species. Each curve represents one PCR amplicon and depicts the change in fluorescence during a continuous

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction, Infection, Amplification, Fluorescence

    Comparison of the universal SYBR green PCR assay with specific TaqMan PCR assays.
    Figure Legend Snippet: Comparison of the universal SYBR green PCR assay with specific TaqMan PCR assays.

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction

    33) Product Images from "Genome-Wide Polysome Profiling Reveals an Inflammation-Responsive Posttranscriptional Operon in Gamma Interferon-Activated Monocytes ▿Genome-Wide Polysome Profiling Reveals an Inflammation-Responsive Posttranscriptional Operon in Gamma Interferon-Activated Monocytes ▿ †"

    Article Title: Genome-Wide Polysome Profiling Reveals an Inflammation-Responsive Posttranscriptional Operon in Gamma Interferon-Activated Monocytes ▿Genome-Wide Polysome Profiling Reveals an Inflammation-Responsive Posttranscriptional Operon in Gamma Interferon-Activated Monocytes ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00824-08

    Validation of the potential target mRNAs by real-time PCR analysis. (A) Total RNAs were isolated from the translationally active polysomal fraction and inactive free fraction after 4 and 18 h of IFN-γ treatment of normal U937 cells. The total mRNAs from unfractionated cell lysates were also isolated at 0, 4, and 18 h of IFN-γ treatment to check the steady-state levels. Total RNAs were subjected to reverse transcription using a TaqMan Reverse Transcription Reagent Kit (Applied Biosystem, Foster City, CA). PCR amplification was carried out using Sybr Green PCR Master Mix (Applied Biosystem, Foster City, CA) and an ABI Thermo Cycler (ABI Prism 7000 SDS). In addition, the authenticity of each primer pair used in real-time PCR was confirmed by classical reverse transcription-PCR and sequencing of the amplified products (100 to 200 bp). The value shown is the relative amount of each mRNA after normalization with GAPDH from triplicates. (B) Status of polysomal and nonpolysomal abundances of two IFN-γ-induced and two noninduced mRNAs upon IFN-γ treatment as a negative control. Total RNAs were isolated from the translationally active polysomal fraction and inactive free fraction after 4 and 18 h of IFN-γ treatment of normal U937 cells. The total mRNAs from unfractionated cell lysates were also isolated at 0, 4, and 18 h of IFN-γ treatment to check the steady-state levels. Total RNAs were subjected to real-time PCR using a TaqMan Reverse Transcription Reagent Kit, Sybr Green PCR Master Mix, and an ABI Thermo Cycler (ABI Prism 7000 SDS). The value shown is the relative amount of each mRNA after normalization with GAPDH from triplicates. (C) The experiments described in panel A above were carried out using L13a-depleted cells expressing shRNA against L13a and control shRNA.
    Figure Legend Snippet: Validation of the potential target mRNAs by real-time PCR analysis. (A) Total RNAs were isolated from the translationally active polysomal fraction and inactive free fraction after 4 and 18 h of IFN-γ treatment of normal U937 cells. The total mRNAs from unfractionated cell lysates were also isolated at 0, 4, and 18 h of IFN-γ treatment to check the steady-state levels. Total RNAs were subjected to reverse transcription using a TaqMan Reverse Transcription Reagent Kit (Applied Biosystem, Foster City, CA). PCR amplification was carried out using Sybr Green PCR Master Mix (Applied Biosystem, Foster City, CA) and an ABI Thermo Cycler (ABI Prism 7000 SDS). In addition, the authenticity of each primer pair used in real-time PCR was confirmed by classical reverse transcription-PCR and sequencing of the amplified products (100 to 200 bp). The value shown is the relative amount of each mRNA after normalization with GAPDH from triplicates. (B) Status of polysomal and nonpolysomal abundances of two IFN-γ-induced and two noninduced mRNAs upon IFN-γ treatment as a negative control. Total RNAs were isolated from the translationally active polysomal fraction and inactive free fraction after 4 and 18 h of IFN-γ treatment of normal U937 cells. The total mRNAs from unfractionated cell lysates were also isolated at 0, 4, and 18 h of IFN-γ treatment to check the steady-state levels. Total RNAs were subjected to real-time PCR using a TaqMan Reverse Transcription Reagent Kit, Sybr Green PCR Master Mix, and an ABI Thermo Cycler (ABI Prism 7000 SDS). The value shown is the relative amount of each mRNA after normalization with GAPDH from triplicates. (C) The experiments described in panel A above were carried out using L13a-depleted cells expressing shRNA against L13a and control shRNA.

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Amplification, SYBR Green Assay, Sequencing, Negative Control, Expressing, shRNA

    34) Product Images from "M-CSF Signals through the MAPK/ERK Pathway via Sp1 to Induce VEGF Production and Induces Angiogenesis In Vivo"

    Article Title: M-CSF Signals through the MAPK/ERK Pathway via Sp1 to Induce VEGF Production and Induces Angiogenesis In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003405

    M-CSF regulates VEGF mRNA transcription through ERK and Sp1. A) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hours in minimal media. The cells were stimulated with 100 ng/ml rhM-CSF for 6 hours followed by isolation of RNA. cDNA was synthesized from total cellular RNA, standardized, and subjected to SYBR Green real-time PCR using primers specific for VEGF-A. The data represents the mean+/−SEM of six individual donors. B) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hrs followed by pre-treatment with either DMSO (vehicle control) or U0126 (10 µM) in minimal media for 30 minutes. The cells were then stimulated with 100 ng/ml rhM-CSF (M-CSF) , (M-CSF+DMSO) , (M-CSF+U0126 10 µM) and analyzed as stated in A. The data represents the mean+/−SEM of three individual donors. C) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hours followed by pre-treatment with either methanol (vehicle control) or mithramycin (200 nM) in minimal media. The cells were then stimulated with 100 ng/ml rhM-CSF (M-CSF) , (M-CSF+MeOH) , (M-CSF+Mith (200 nM)) and analyzed as in A. The data represents the mean+/−SEM of four individual donors.
    Figure Legend Snippet: M-CSF regulates VEGF mRNA transcription through ERK and Sp1. A) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hours in minimal media. The cells were stimulated with 100 ng/ml rhM-CSF for 6 hours followed by isolation of RNA. cDNA was synthesized from total cellular RNA, standardized, and subjected to SYBR Green real-time PCR using primers specific for VEGF-A. The data represents the mean+/−SEM of six individual donors. B) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hrs followed by pre-treatment with either DMSO (vehicle control) or U0126 (10 µM) in minimal media for 30 minutes. The cells were then stimulated with 100 ng/ml rhM-CSF (M-CSF) , (M-CSF+DMSO) , (M-CSF+U0126 10 µM) and analyzed as stated in A. The data represents the mean+/−SEM of three individual donors. C) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hours followed by pre-treatment with either methanol (vehicle control) or mithramycin (200 nM) in minimal media. The cells were then stimulated with 100 ng/ml rhM-CSF (M-CSF) , (M-CSF+MeOH) , (M-CSF+Mith (200 nM)) and analyzed as in A. The data represents the mean+/−SEM of four individual donors.

    Techniques Used: Isolation, Cell Culture, Synthesized, SYBR Green Assay, Real-time Polymerase Chain Reaction

    35) Product Images from "Biochemical Properties of Purified Human Retinol Dehydrogenase 12 (RDH12): Catalytic Efficiency toward Retinoids and C9 Aldehydes and Effects of Cellular Retinol-Binding Protein Type I (CRBPI) and Cellular Retinaldehyde-Binding Protein (CRALBP) on the Oxidation and Reduction of Retinoids †"

    Article Title: Biochemical Properties of Purified Human Retinol Dehydrogenase 12 (RDH12): Catalytic Efficiency toward Retinoids and C9 Aldehydes and Effects of Cellular Retinol-Binding Protein Type I (CRBPI) and Cellular Retinaldehyde-Binding Protein (CRALBP) on the Oxidation and Reduction of Retinoids †

    Journal: Biochemistry

    doi: 10.1021/bi050226k

    Human tissue expression profile of RDH12 and CRBPI. SYBR green real-time quantitative RT-PCR was carried out using 200 pg of cDNA from the indicated tissues or 12.5 pg of retina cDNA and primers specific for human RDH12, CRBPI, and GAPDH. RDH12 and CRBPI
    Figure Legend Snippet: Human tissue expression profile of RDH12 and CRBPI. SYBR green real-time quantitative RT-PCR was carried out using 200 pg of cDNA from the indicated tissues or 12.5 pg of retina cDNA and primers specific for human RDH12, CRBPI, and GAPDH. RDH12 and CRBPI

    Techniques Used: Expressing, SYBR Green Assay, Quantitative RT-PCR

    36) Product Images from "An in vitro model of Mycobacterium leprae induced granuloma formation"

    Article Title: An in vitro model of Mycobacterium leprae induced granuloma formation

    Journal: BMC Infectious Diseases

    doi: 10.1186/1471-2334-13-279

    Determination of mRNA level of cytokines by real-time PCR. mRNA expression of IFN-γ, IL-2, TNF-α, IL-12, and IL-1β in the cells was evaluated by using specific primers, SYBR Green PCR Master Mix, and a StepOnePlus (Applied Biosystems). Cells were collected on days 1, 3 and 9 after co-culture with or without PBMCs. The y-axis shows the relative quantitative values calculated from comparative Ct values normalized to the control (actin used as calibrator). The standard deviation of the ΔCt value was determined (Applied Biosystems). Representative data from a single donor of three individual experiments are shown.
    Figure Legend Snippet: Determination of mRNA level of cytokines by real-time PCR. mRNA expression of IFN-γ, IL-2, TNF-α, IL-12, and IL-1β in the cells was evaluated by using specific primers, SYBR Green PCR Master Mix, and a StepOnePlus (Applied Biosystems). Cells were collected on days 1, 3 and 9 after co-culture with or without PBMCs. The y-axis shows the relative quantitative values calculated from comparative Ct values normalized to the control (actin used as calibrator). The standard deviation of the ΔCt value was determined (Applied Biosystems). Representative data from a single donor of three individual experiments are shown.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, SYBR Green Assay, Polymerase Chain Reaction, Co-Culture Assay, Standard Deviation

    37) Product Images from "Impaired Wnt Signaling in Embryonal Rhabdomyosarcoma Cells from p53/c-fos Double Mutant Mice"

    Article Title: Impaired Wnt Signaling in Embryonal Rhabdomyosarcoma Cells from p53/c-fos Double Mutant Mice

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2010.091195

    SYBR Green Real-Time RT-PCR
    Figure Legend Snippet: SYBR Green Real-Time RT-PCR

    Techniques Used: SYBR Green Assay, Quantitative RT-PCR

    38) Product Images from "Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells"

    Article Title: Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

    Journal:

    doi: 10.3748/wjg.14.1790

    Quantitative real-time PCR using SYBR green I
    Figure Legend Snippet: Quantitative real-time PCR using SYBR green I

    Techniques Used: Real-time Polymerase Chain Reaction, SYBR Green Assay

    39) Product Images from "Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications"

    Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications

    Journal: The FASEB Journal

    doi: 10.1096/fj.08-117697

    SYBR Green-based real-time PCR
    Figure Legend Snippet: SYBR Green-based real-time PCR

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction

    40) Product Images from "Hypomethylation and expression of BEX2, IGSF4 and TIMP3 indicative of MLL translocations in Acute Myeloid Leukemia"

    Article Title: Hypomethylation and expression of BEX2, IGSF4 and TIMP3 indicative of MLL translocations in Acute Myeloid Leukemia

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-8-86

    TSG methylation predictive for classification of acute leukemia cell lines . Methylation was assessed by MS-MLPA (see also Additional file 1 ) or by DNA digestion with methylation-sensitive Hha I plus promoter-specific quantitative real-time PCR (for analysis of BEX2 ). Green: methylation level ≥ 10%; yellow: methylation level
    Figure Legend Snippet: TSG methylation predictive for classification of acute leukemia cell lines . Methylation was assessed by MS-MLPA (see also Additional file 1 ) or by DNA digestion with methylation-sensitive Hha I plus promoter-specific quantitative real-time PCR (for analysis of BEX2 ). Green: methylation level ≥ 10%; yellow: methylation level

    Techniques Used: Methylation, Mass Spectrometry, Multiplex Ligation-dependent Probe Amplification, Real-time Polymerase Chain Reaction

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Quantification of Multiple Gene Expression in Individual Cells
    Article Snippet: .. Real-time quantitative PCR was performed by adding 10 μL of 2× SYBR Green PCR Master Mix (Applied Biosystems) to each well containing 4 μL of template and 6 μL of a primer mix with 0.25 μM of each specific primer (see Supplemental materials I and II) in a 20-μL reaction volume using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). .. After a denaturation step at 95°C for 10 min, the cycle profile used was 30 sec at 95°C, 30 sec at 60°C, and 45 sec at 72°C for 60 cycles of amplification.

    Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection
    Article Snippet: .. Quantitative PCR was performed using SYBR Green PCR Master Mix (#4309155, Applied Biosystems, Foster City, CA) with 5 μl of immunoprecipitated material, 0.2 μM of primer [HIV-1 LTR (−69 − +175) Forward 5′-CTGGGCGGGACTGGGGAG-3′ and Reverse 5′-TCACACAACAGACGGGCACAC-3′]. .. The antibodies used for immunoprecipitation were as follows: total RNAP II CTD (ab817, Abcam), pS10-H3 (Novex, Life Technologies), BRCA1 (sc-642, Santa Cruz), p-BRCA1 S1423 (sc-101647, Santa, Cruz), Sp1 (5931, Cell Signaling), (IgG (sc-2027, Santa Cruz) or V5 (AbD Serotec, Oxford, UK).

    Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection
    Article Snippet: .. Quantitative PCR was performed with SYBR Green PCR Master Mix Applied Biosystems). ..

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: .. 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix. .. For the SYBR green PCR assay, the 20 μl PCR mix reaction mix was comprised of 5 μl of cDNA and 15 μl of master mix prepared using 1 μl of the 10 pmol forward and reverse primers, 10 μl SYBR® Green I and 3 μl of nuclease free water.

    Amplification:

    Article Title: CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use
    Article Snippet: .. An aliquot of 5 μL of the RT reaction was amplified in duplicate in a final volume of 30 μL of SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA, USA). .. The sample target RNA copy numbers were calculated using SDS 1.9 Software (Applied Biosystems, Foster City, CA, USA).

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells
    Article Snippet: .. The resultant cDNA was amplified using Sybr Green PCR master mix (Gibco/Life Technologies) on a StepOnePlus system according to manufacturer’s protocol. ..

    In Vitro:

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: .. 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix. .. For the SYBR green PCR assay, the 20 μl PCR mix reaction mix was comprised of 5 μl of cDNA and 15 μl of master mix prepared using 1 μl of the 10 pmol forward and reverse primers, 10 μl SYBR® Green I and 3 μl of nuclease free water.

    Sequencing:

    Article Title: Quantification of Multiple Gene Expression in Individual Cells
    Article Snippet: .. Real-time quantitative PCR was performed by adding 10 μL of 2× SYBR Green PCR Master Mix (Applied Biosystems) to each well containing 4 μL of template and 6 μL of a primer mix with 0.25 μM of each specific primer (see Supplemental materials I and II) in a 20-μL reaction volume using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). .. After a denaturation step at 95°C for 10 min, the cycle profile used was 30 sec at 95°C, 30 sec at 60°C, and 45 sec at 72°C for 60 cycles of amplification.

    Quantitative RT-PCR:

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: .. 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix. .. For the SYBR green PCR assay, the 20 μl PCR mix reaction mix was comprised of 5 μl of cDNA and 15 μl of master mix prepared using 1 μl of the 10 pmol forward and reverse primers, 10 μl SYBR® Green I and 3 μl of nuclease free water.

    SYBR Green Assay:

    Article Title: Quantification of Multiple Gene Expression in Individual Cells
    Article Snippet: .. Real-time quantitative PCR was performed by adding 10 μL of 2× SYBR Green PCR Master Mix (Applied Biosystems) to each well containing 4 μL of template and 6 μL of a primer mix with 0.25 μM of each specific primer (see Supplemental materials I and II) in a 20-μL reaction volume using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). .. After a denaturation step at 95°C for 10 min, the cycle profile used was 30 sec at 95°C, 30 sec at 60°C, and 45 sec at 72°C for 60 cycles of amplification.

    Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection
    Article Snippet: .. Quantitative PCR was performed using SYBR Green PCR Master Mix (#4309155, Applied Biosystems, Foster City, CA) with 5 μl of immunoprecipitated material, 0.2 μM of primer [HIV-1 LTR (−69 − +175) Forward 5′-CTGGGCGGGACTGGGGAG-3′ and Reverse 5′-TCACACAACAGACGGGCACAC-3′]. .. The antibodies used for immunoprecipitation were as follows: total RNAP II CTD (ab817, Abcam), pS10-H3 (Novex, Life Technologies), BRCA1 (sc-642, Santa Cruz), p-BRCA1 S1423 (sc-101647, Santa, Cruz), Sp1 (5931, Cell Signaling), (IgG (sc-2027, Santa Cruz) or V5 (AbD Serotec, Oxford, UK).

    Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection
    Article Snippet: .. Quantitative PCR was performed with SYBR Green PCR Master Mix Applied Biosystems). ..

    Article Title: CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use
    Article Snippet: .. An aliquot of 5 μL of the RT reaction was amplified in duplicate in a final volume of 30 μL of SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA, USA). .. The sample target RNA copy numbers were calculated using SDS 1.9 Software (Applied Biosystems, Foster City, CA, USA).

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells
    Article Snippet: .. The resultant cDNA was amplified using Sybr Green PCR master mix (Gibco/Life Technologies) on a StepOnePlus system according to manufacturer’s protocol. ..

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: .. 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix. .. For the SYBR green PCR assay, the 20 μl PCR mix reaction mix was comprised of 5 μl of cDNA and 15 μl of master mix prepared using 1 μl of the 10 pmol forward and reverse primers, 10 μl SYBR® Green I and 3 μl of nuclease free water.

    Article Title: A Myc-microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes
    Article Snippet: .. PCR was performed using the SYBR GREEN PCR Master Mix from Applied Biosystems. .. The target-gene messenger RNA (mRNA) expression was normalized to the expression of GAPDH, and relative mRNA fold changes were calculated by the ΔΔCt method.

    Article Title: hCAF1/CNOT7 regulates interferon signalling by targeting STAT1
    Article Snippet: .. After DNA purification, qPCRs were performed in triplicate in a 96-well optical reaction plate using SYBR Green PCR Master Mix (Life Technology). .. The –ΔΔCt values for each locus were calculated with respect to the ChIP input DNA, normalized to a reference locus (3′ downstream region of the GAPDH gene).

    Immunoprecipitation:

    Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection
    Article Snippet: .. Quantitative PCR was performed using SYBR Green PCR Master Mix (#4309155, Applied Biosystems, Foster City, CA) with 5 μl of immunoprecipitated material, 0.2 μM of primer [HIV-1 LTR (−69 − +175) Forward 5′-CTGGGCGGGACTGGGGAG-3′ and Reverse 5′-TCACACAACAGACGGGCACAC-3′]. .. The antibodies used for immunoprecipitation were as follows: total RNAP II CTD (ab817, Abcam), pS10-H3 (Novex, Life Technologies), BRCA1 (sc-642, Santa Cruz), p-BRCA1 S1423 (sc-101647, Santa, Cruz), Sp1 (5931, Cell Signaling), (IgG (sc-2027, Santa Cruz) or V5 (AbD Serotec, Oxford, UK).

    DNA Purification:

    Article Title: hCAF1/CNOT7 regulates interferon signalling by targeting STAT1
    Article Snippet: .. After DNA purification, qPCRs were performed in triplicate in a 96-well optical reaction plate using SYBR Green PCR Master Mix (Life Technology). .. The –ΔΔCt values for each locus were calculated with respect to the ChIP input DNA, normalized to a reference locus (3′ downstream region of the GAPDH gene).

    Polymerase Chain Reaction:

    Article Title: Quantification of Multiple Gene Expression in Individual Cells
    Article Snippet: .. Real-time quantitative PCR was performed by adding 10 μL of 2× SYBR Green PCR Master Mix (Applied Biosystems) to each well containing 4 μL of template and 6 μL of a primer mix with 0.25 μM of each specific primer (see Supplemental materials I and II) in a 20-μL reaction volume using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). .. After a denaturation step at 95°C for 10 min, the cycle profile used was 30 sec at 95°C, 30 sec at 60°C, and 45 sec at 72°C for 60 cycles of amplification.

    Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection
    Article Snippet: .. Quantitative PCR was performed using SYBR Green PCR Master Mix (#4309155, Applied Biosystems, Foster City, CA) with 5 μl of immunoprecipitated material, 0.2 μM of primer [HIV-1 LTR (−69 − +175) Forward 5′-CTGGGCGGGACTGGGGAG-3′ and Reverse 5′-TCACACAACAGACGGGCACAC-3′]. .. The antibodies used for immunoprecipitation were as follows: total RNAP II CTD (ab817, Abcam), pS10-H3 (Novex, Life Technologies), BRCA1 (sc-642, Santa Cruz), p-BRCA1 S1423 (sc-101647, Santa, Cruz), Sp1 (5931, Cell Signaling), (IgG (sc-2027, Santa Cruz) or V5 (AbD Serotec, Oxford, UK).

    Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection
    Article Snippet: .. Quantitative PCR was performed with SYBR Green PCR Master Mix Applied Biosystems). ..

    Article Title: CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use
    Article Snippet: .. An aliquot of 5 μL of the RT reaction was amplified in duplicate in a final volume of 30 μL of SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA, USA). .. The sample target RNA copy numbers were calculated using SDS 1.9 Software (Applied Biosystems, Foster City, CA, USA).

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells
    Article Snippet: .. The resultant cDNA was amplified using Sybr Green PCR master mix (Gibco/Life Technologies) on a StepOnePlus system according to manufacturer’s protocol. ..

    Article Title: A Myc-microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes
    Article Snippet: .. PCR was performed using the SYBR GREEN PCR Master Mix from Applied Biosystems. .. The target-gene messenger RNA (mRNA) expression was normalized to the expression of GAPDH, and relative mRNA fold changes were calculated by the ΔΔCt method.

    Article Title: hCAF1/CNOT7 regulates interferon signalling by targeting STAT1
    Article Snippet: .. After DNA purification, qPCRs were performed in triplicate in a 96-well optical reaction plate using SYBR Green PCR Master Mix (Life Technology). .. The –ΔΔCt values for each locus were calculated with respect to the ChIP input DNA, normalized to a reference locus (3′ downstream region of the GAPDH gene).

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    Thermo Fisher sybr green i
    Correlation of detection of virus by Bio-Plex and RT-qPCR. Correlation between qPCR (Ct value) and Bio-Plex (MFI) detection of in-vitro grown ISAV standard curve by (A) <t>SYBR</t> <t>GREEN</t> I and (B)TaqMan; Correlation of detection of copy number of ISAV in plasma samples by (C) SYBR Green I (n = 13) and (D) TaqMan chemistries (n = 8).
    Sybr Green I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 13018 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fast sybr green master mix
    HuR associates with SCN5A mRNA (A) Schematic representation of the SCN5A mRNA ARE sites in its 3′-UTR. The length of the mRNA is indicated by a kilo base (kb)-scale. (B) HuR binds to the SCN5A mRNA. After IP of RNA-protein complexes from cardiomyocytes using either anti-HuR antibody (Anti-HuR) or control IgG, RNA was isolated, and then, the cDNA was synthesized in a presence or absence of the reverse transcription enzyme. Two sets of primers to different regions of SCN5A were used to amplify SCN5A fragments. The PCR products were visualized with <t>SYBR</t> ™ Safe DNA Gel Stain in agarose gels. (C) <t>qRT-PCR</t> was performed to confirm the results in (B).
    Fast Sybr Green Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher real time quantitative pcr analysis
    Confirmation of RNA and protein expression levels by real-time quantitative <t>PCR</t> and western blotting analysis (n = 3, per treatment type). (A) The expression of LASP1 and ferritin light and heavy chain mRNAs relative to that of GAPDH, the internal control, as detected by real-time PCR. (B) Expression of LASP1 and ferritin light and heavy chains, as assessed by western blotting analysis. The signal corresponding to the protein bands was quantified using densitometric scanning and the relative protein abundance was determined after normalization with the levels of β-actin protein. Data are presented as mean ± SD and compared using the t -test for independent samples. * p
    Real Time Quantitative Pcr Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 266 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Correlation of detection of virus by Bio-Plex and RT-qPCR. Correlation between qPCR (Ct value) and Bio-Plex (MFI) detection of in-vitro grown ISAV standard curve by (A) SYBR GREEN I and (B)TaqMan; Correlation of detection of copy number of ISAV in plasma samples by (C) SYBR Green I (n = 13) and (D) TaqMan chemistries (n = 8).

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0159155

    Figure Lengend Snippet: Correlation of detection of virus by Bio-Plex and RT-qPCR. Correlation between qPCR (Ct value) and Bio-Plex (MFI) detection of in-vitro grown ISAV standard curve by (A) SYBR GREEN I and (B)TaqMan; Correlation of detection of copy number of ISAV in plasma samples by (C) SYBR Green I (n = 13) and (D) TaqMan chemistries (n = 8).

    Article Snippet: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix.

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0159155

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

    Article Snippet: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix.

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

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

    Journal: International Journal of Clinical and Experimental Pathology

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

    doi:

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

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

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

    HuR associates with SCN5A mRNA (A) Schematic representation of the SCN5A mRNA ARE sites in its 3′-UTR. The length of the mRNA is indicated by a kilo base (kb)-scale. (B) HuR binds to the SCN5A mRNA. After IP of RNA-protein complexes from cardiomyocytes using either anti-HuR antibody (Anti-HuR) or control IgG, RNA was isolated, and then, the cDNA was synthesized in a presence or absence of the reverse transcription enzyme. Two sets of primers to different regions of SCN5A were used to amplify SCN5A fragments. The PCR products were visualized with SYBR ™ Safe DNA Gel Stain in agarose gels. (C) qRT-PCR was performed to confirm the results in (B).

    Journal: Heart rhythm

    Article Title: HuR-mediated SCN5A mRNA stability reduces arrhythmic risk in heart failure

    doi: 10.1016/j.hrthm.2018.02.018

    Figure Lengend Snippet: HuR associates with SCN5A mRNA (A) Schematic representation of the SCN5A mRNA ARE sites in its 3′-UTR. The length of the mRNA is indicated by a kilo base (kb)-scale. (B) HuR binds to the SCN5A mRNA. After IP of RNA-protein complexes from cardiomyocytes using either anti-HuR antibody (Anti-HuR) or control IgG, RNA was isolated, and then, the cDNA was synthesized in a presence or absence of the reverse transcription enzyme. Two sets of primers to different regions of SCN5A were used to amplify SCN5A fragments. The PCR products were visualized with SYBR ™ Safe DNA Gel Stain in agarose gels. (C) qRT-PCR was performed to confirm the results in (B).

    Article Snippet: Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) was carried out using gene-specific primers, Fast SYBR® Green Master Mix and 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Isolation, Synthesized, Polymerase Chain Reaction, Staining, Quantitative RT-PCR

    Confirmation of RNA and protein expression levels by real-time quantitative PCR and western blotting analysis (n = 3, per treatment type). (A) The expression of LASP1 and ferritin light and heavy chain mRNAs relative to that of GAPDH, the internal control, as detected by real-time PCR. (B) Expression of LASP1 and ferritin light and heavy chains, as assessed by western blotting analysis. The signal corresponding to the protein bands was quantified using densitometric scanning and the relative protein abundance was determined after normalization with the levels of β-actin protein. Data are presented as mean ± SD and compared using the t -test for independent samples. * p

    Journal: Proteome Science

    Article Title: Proteomic analysis on effectors involved in BMP-2-induced osteogenic differentiation of beagle bone marrow mesenchymal stem cells

    doi: 10.1186/1477-5956-12-13

    Figure Lengend Snippet: Confirmation of RNA and protein expression levels by real-time quantitative PCR and western blotting analysis (n = 3, per treatment type). (A) The expression of LASP1 and ferritin light and heavy chain mRNAs relative to that of GAPDH, the internal control, as detected by real-time PCR. (B) Expression of LASP1 and ferritin light and heavy chains, as assessed by western blotting analysis. The signal corresponding to the protein bands was quantified using densitometric scanning and the relative protein abundance was determined after normalization with the levels of β-actin protein. Data are presented as mean ± SD and compared using the t -test for independent samples. * p

    Article Snippet: The cDNA was then used as the template for real-time quantitative PCR analysis (Maxima SYBR Green/ROX qPCR Master Mix; Thermo Scientific, Pittsburgh, PA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot