Hiscript 2 q rt supermix for qpcr gdna wiper 1st strand cdna synthesis kit

Following the manufacturer’s instructions, Trizole reagent was used to extract total RNA from individual treatment leaf samples. With the help of agarose gel electrophoresis, a NanoPhotometer^® spectrophotometer (Implen, Westlake Village, CA, USA), the extracted RNA’ purity and quality were examined. The manufacturer protocol of Vazyme HiScript II Q RT SuperMix for qPCR (+gDNA wiper) 1st strand cDNA synthesis kit (Vazyme, Nanjing, China) was followed to reverse-transcribe the extracted RNA, for complementary DNA (cDNA) synthesis. For qRT-PCR (quantitative real-time PCR) analysis, cDNA was used as templates. The Roche FastStart Essential DNA Green Master kit (Roche, Pleasanton, CA, USA) was employed in an Mx3000 P qPCR system (Agilent Technologies, Santa Clara, CA, USA), and 96-well plates were used to perform qRT-PCR. Supplementary Table S1 provides the detail of primers followed in this study Jahan et al. [43 (link)], and Actin was used as a reference gene. The formula of Livak and Schmittgen [44 (link)], i.e., 2^−ΔΔCt was used to calculate the changes in relative gene expression. Three biological replications were performed for each treatment, and three technical replicates were carried out for each biological replicate.

After 7 days of CS treatment, total RNA was extracted from individual treatment leaf samples using Trizole reagent, following the manufacturer’s instructions. The quality and purity of the extracted RNA was examined using agarose gel electrophoresis, with a Nanodrop 2000 spectrophotometer^® (Thermo, Deutshland, Germany). For the complementary DNA (cDNA) synthesis, extracted RNA was reverse-transcribed using the Vazyme HiScript II QRT SuperMix for qPCR (+gDNA wiper) 1st-strand cDNA synthesis kit (Vazyme, Nanjing, China), following the manufacturer’s protocol, and the cDNA was used as a template for the quantitative real-time PCR (qRT PCR) analysis. The qRT PCR was performed with 96-well plates using an Mx3000 P qPCR system (Agilent Technologies, Santa Clara, CA, USA) and the Roche FastStart Essential DNA Green Master kit (Roche, Indianapolis, IN, USA). The primers used in this experiment are given in Table S1. The actin gene was used as an internal reference gene. The values of relative expression level changes were calculated using the formula 2^−ΔΔCt [43 (link)].