Quantitative Real-Time PCR Analysis

A qRT-PCR analysis, using 2 × ChamQ™ SYBR qPCR Master Mix (Vazyme#Q311, Nanjing, China), was performed on an iQ^TM 5 Connect Real-Time System (BIO-RAD, Hercules, CA, USA) [29 (link)]. The real-time quantitative PCR reaction system (20 μL) consisted of 2 μg/μL of cDNA, 0.5 μL of each primer (10 μM), 10 μL of 2 × Premix, and the appropriate volume of double-distilled H₂O. Then, the expression analysis of three replicates was performed by the CFX96 Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA). The thermal cycling conditions were an initial denaturation at 94 °C for 2 min, followed by 42 cycles of amplification (denaturation at 94 °C for 15 s and annealing/extension at 60 °C for 30 s). VviActin was used as the internal standard gene. The relative expression levels of genes were calculated using the 2^−ΔCT method [27 (link)].