Quantitative Real-Time PCR Gene Expression Analysis

Total RNA from cells was isolated using a GenElute™ Total RNA Purification Kit (Sigma). First, strand cDNA was reverse transcribed using iScript™ Advanced cDNA Synthesis Kit (Bio-Rad). Specific primers (Table 1, designed using NCBI Primer Blast and Primer 3.0 InPut software) were synthesized by Sigma-Aldrich. Using the cDNA samples, quantitative PCR assays (in triplicate) were performed using SsoAdvanced™ Universal SYBR^® Green Supermix (Bio-Rad) and a CFX PCR system (Bio-Rad). After being normalized to the expression of internal housekeeping gene cyclophilin A (CycA) [13 (link),28 (link),30 (link),31 (link)], the relative expression of genes of interest was calculated with the comparative 2^−ΔΔCt method.