A sample of cDNA, derived from equal masses of RNA from each volunteer, was amplified in duplicate (with an additional independent “no reverse transcriptase-treated” control) using the USB VeriQuest Fast SYBR Green qPCR Master Mix (Affymetrix, Cleveland, OH) and custom-designed, intron-spanning PCR primers (Supplemental Table S1 and Figure S1 ). Cycling conditions were modified from the manufacturer's suggested protocol to establish linearity of transcript detection (Supplemental Figure S2A,B ). Relative quantification of gene expression was performed using the ΔΔCT method41 (link), and the specificity of each amplicon was verified by gel electrophoresis and melt curve tests (e.g., Supplemental Fig. S2C ).
Usb veriquest fast sybr green qpcr master mix
Manufactured by Thermo Fisher Scientific
The USB VeriQuest Fast SYBR Green qPCR Master Mix is a reagent designed for quantitative real-time PCR (qPCR) analysis. It contains all the necessary components, including SYBR Green I dye, for the amplification and detection of DNA sequences.
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2 protocols using usb veriquest fast sybr green qpcr master mix
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Quantitative RT-PCR for Gene Expression
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Quantitative RT-PCR for Gene Expression
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A sample of cDNA, derived from equal masses of RNA from each volunteer, was amplified in duplicate (with an additional independent “no reverse transcriptase-treated” control) using the USB VeriQuest Fast SYBR Green qPCR Master Mix (Affymetrix, Cleveland, OH) and custom-designed, intron-spanning PCR primers (Supplemental Table S1 and Figure S1 ). Cycling conditions were modified from the manufacturer's suggested protocol to establish linearity of transcript detection (Supplemental Figure S2A,B ). Relative quantification of gene expression was performed using the ΔΔCT method41 (link), and the specificity of each amplicon was verified by gel electrophoresis and melt curve tests (e.g., Supplemental Fig. S2C ).
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