RNA was harvested from tissue or dissociated cells using TRIzol (Invitrogen), and cDNA was prepared for PCR using standard methods (22 (link)). Real-time qPCR was performed using gene-specific primers (Supplementary Table S1 ) and either Power Sybr Green Master Mix Reagent (Life Technologies) or iTaq Sybr Green (Bio-Rad), with either a MiniOpticon Real-Time PCR machine or CFX Connect Real-Time PCR machine (Bio-Rad). Data were analyzed according to the ΜCt method (2−ΔΔt), where ΔCt is the difference between the gene of interest and GAPDH control Ct value, and data were normalized to the average of all vehicle-treated samples as in refs. 21 (link), 22 (link).
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Real-Time qPCR for Gene Expression Analysis
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Corresponding Organization : Washington University in St. Louis
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Variable analysis
independent variables
- Treatment condition (e.g., vehicle vs. drug treatment)
- Gene expression levels measured by real-time qPCR
- GAPDH control as reference gene for normalization of gene expression data
- Positive control: None specified
- Negative control: None specified
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