Gene expression analysis by qRT-PCR

For expression validation of differentially expressed genes, special primers were designed for 15 differentially expressed genes, randomly selected in different time points using Primer Premier 5.0 software (Supplementary Table S2). The specificity of each primer set was checked by sequencing PCR products and efficiencies of the different primer sets were similar. Total RNA used as template for reverse transcriptase reactions was initially treated with DNase I Amplification Grade enzyme (Invitrogen), an aliquot of treated RNA was used in qPCR to rule out DNA contamination. cDNA synthesis was done using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) and random hexamers and oligo(dT) primers. qPCR reaction were performed in a volume of 20 μL containing 10 μL 2 × All-in-One^TM (GeneCopoeia, Los Angeles, USA) qPCR Mix, 2 μL cDNA, 1 μL of each 4 μM primer, and 6 μL RNase-free sterile water. To normalize the relative expression of selected genes, GAPDH gene was used as reference. qPCR was performed using in an iQ5 Real Time PCR Detection System (Bio-Rad, Hercules, USA). PCR reactions were performed at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 20 s. Melting curve analysis was conducted for each reaction to confirm the specificity of the reaction, all the cDNA samples were analyzed in triplicate. Relative expression levels of candidate genes were calculated using 2^−ΔΔCt method⁵⁴.