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 (link)]. The gene expression data were further visualized using the software MeV viewer (http://www.tm4.org ).
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