RNA Extraction, cDNA Synthesis, and RT-qPCR Analysis

RNA isolation was achieved by TRIZOL and the RNA extracts were treated with AMBION DNA-free to remove DNA impurities. The iSCRIPT cDNA synthesis kit was used to synthesise 300ng of cDNA whereas KAPA Taq PCR was used for reverse transcription PCR (RT-PCR). The real time PCR was achieved by KAPA SYBR FAST qPCR Master Mix (2X) kit from Kapa Biosystems according to the manufacturers’ guidelines and the primer sequences for performing both RT-PCR and Real-Time PCR are provided in S3 Table [51 (link)–67 (link), 70 (link)–76 (link)]. For each gene, the average C(t) value was determined and was normalised to housekeeping (GAPDH) mRNA levels. Unpaired t-test (two-tailed) was used to calculate statistical significance.