Quantitative RT-PCR Analysis of Gene Expression

One microgram of DNase treated total RNA was used for first strand cDNA synthesis using Maxima H Minus M-MuLV reverse transcriptase (Thermo Scientific, USA). A 5-fold dilution of cDNA samples was done with deionized water (Sigma, USA) to be used in qRT-PCR reactions. qRT-PCR was carried out using 2X Brilliant III SYBR® Green Q PCR (Agilent Technologies, USA) on a Stratagene Mx3000P (Agilent, USA) with cycling conditions and replications as described in Tiwari et al.^{20 (link)}. The amount of transcript accumulated for each target gene normalized to the reference gene Actin. The qRT- PCR primers were designed using the IDT Primer Quest software with default parameters: Tm = 59–62 °C; GC = 45–55%; amplicon size = 100–150 bp (Table S1). The heatmap for expression profiles of DEGs was generated using the MEV4 software package^{21 (link)}.