2.6. Expression analysis using quantitative RT-PCR

Total RNA isolation and cDNA synthesis were performed as explained in above section. We have used diluted cDNA (100 ng μL⁻¹) for qRT-PCR reactions. Oligo’s for the qRT-PCR was designed using Genefisher2 software. We selected heat-responsive transcription factors - HSFA6e (acc. no. {"type":"entrez-nucleotide","attrs":{"text":"KU291394.1","term_id":"1026817960","term_text":"KU291394.1"}}KU291394.1), WRKY (acc. no. {"type":"entrez-nucleotide","attrs":{"text":"KU562861.1","term_id":"1128621848","term_text":"KU562861.1"}}KU562861.1), HSPs - HSP17 (acc. no. {"type":"entrez-nucleotide","attrs":{"text":"JN572711.1","term_id":"345462628","term_text":"JN572711.1"}}JN572711.1), HSP26 (acc. no. {"type":"entrez-nucleotide","attrs":{"text":"AF097659.1","term_id":"4028572","term_text":"AF097659.1"}}AF097659.1)], antioxidant enzymes – SOD (acc. no. {"type":"entrez-nucleotide","attrs":{"text":"AF092524.1","term_id":"3676838","term_text":"AF092524.1"}}AF092524.1), POX (acc. no. {"type":"entrez-nucleotide","attrs":{"text":"AF005087.1","term_id":"6996624","term_text":"AF005087.1"}}AF005087.1) and starch biosynthesis pathway linked genes – ADP-glucose pyrophosphorylase (large subunit; acc. no. {"type":"entrez-nucleotide","attrs":{"text":"KC347594.1","term_id":"469952289","term_text":"KC347594.1"}}KC347594.1), soluble starch synthase gene (acc. no. {"type":"entrez-nucleotide","attrs":{"text":"KJ854903.1","term_id":"673575963","term_text":"KJ854903.1"}}KJ854903.1) for the expression analysis (Table S3). The samples were used in triplicates for the expression analysis. The expression was performed following the protocol as mentioned in Kumar et al. [20]. We have used β-actin gene (acc. no. {"type":"entrez-nucleotide","attrs":{"text":"AB181991.1","term_id":"48927617","term_text":"AB181991.1"}}AB181991.1) for normalizing the C_t value. Further, Pfaffl method [24] was used for calculating the relative fold expression of the genes.