Quantitative Gene Expression Analysis in Plants

Real-time PCR was performed using Roche LightCycler 480II, with 20 μL reaction mixtures, containing 10 μL of SYBR Premix Ex TaqTM (2×), 1 μL of cDNA, 0.3 μL of upstream and downstream primers (10 μmoL/L; Table S1), and 8.4 μL of ddH₂O, with three replicates per sample. The relative expression levels of the target genes (AtHSFA2, AtHSP18.2, AtHSP70, and VvDREB2c) were calculated using geNorm [51 (link)], based on a method derived from algorithms outlined using VvEF1γand VvGAPDH as the reference genes in grape [52 (link)] and AtUBQ10 and AtACTIN2 as the reference genes in Arabidopsis [53 (link)]. The primers of AtHSFA2, AtHSP18.2, AtHSP70, and AtHSP21 were from Ikeda et al. [54 (link)]. The primers of other genes that were designed and met the following conditions: the slope of standard curve was maintained at approximately −3.4 and the efficiencies of amplification between >90% and <110%.