Validating Differential Gene Expression by qRT-PCR

Six up-regulated and four down-regulated genes were selected to validate the microarray data by qRT-PCR. Primers were designed using the tool primer3 based on the sequence of each differentially expressed probe accessible on the Affymetrix website (Table S3). RNA was isolated as previously described, treated with RNase Free DNase Qiagen (Cat #79254) and cleaned with Qiagen RNeasy Mini Kit (P/N 74104). cDNA was synthesized from 1 μg RNA using SuperScriptII Platinum Two-Step qRT-PCR kit (Invitrogen cat # 11735). Quantitative real time PCR (qRT-PCR) was performed in a BioRad iCycler using SYBRGreenER™ qPCR SuperMix (Invitrogen cat# 11761) following manufacturer's instructions. Wheat actin gene was used as internal reference to normalize Ct-values obtained for each gene. qRT-PCR data was analyzed based on Dussault and Pouliot (2006 (link)) method.