Quantitative real-time PCR

The qPCR experiments were carried out using SYBR Green Supermix (BioRad) in an iQ5 real-time thermal cycler (Bio-Rad, USA). Each 25μl reaction comprised 4μl cDNA template (2.5μg/μl), 12.5μl SYBR Green Supermix (Bio-Rad, USA), 0.4μl of each primer (10μM) and 0.7μl of sterile distilled water. Thermal cycling started with a denaturation step at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 15s and annealing at the respective temperature for each gene (Table 1) for 30s. Each set of reactions included a negative control with no template. The amplification efficiency for each gene of interest was determined using the LinRegPCR version 2013.0. Dissociation curves (S2 Fig) and agarose gel electrophoresis were used to analyze non-specific PCR products. Three biological replicates and two technical replicates were used for each sample. Relative gene expression (fold change) was calculated according to Hellemans et al., (2007) [47]. The gene expression data were further visualized using the software MeV viewer (http://www.tm4.org).