Sybr green premix ex taq 2 quantitative pcr system

Total RNA was isolated from the four to six euphylla stages of capsicum 6421 using the RNAiso Plus reagent (TaKaRa, Dalian, China) and then treated with RNase-free DNase I (Promega). Subsequently, 0.5 μg RNA was used for first-strand cDNA synthesis using a HiScript II 1st Strand cDNA Synthesis kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. qPCR was performed using LightCycler 96 (Roche, Basel, Switzerland) with the SYBR Green Premix Ex Taq™ II quantitative PCR system (TaKaRa), and the primers of eight subclasses of genes are listed in Table S5. At least three biological replicates were included. Briefly, after an initial denaturation step at 95 °C for 10 min, the amplifications were carried out with 40 cycles at a melting temperature of 95 °C for 15 s and an annealing temperature of 60 °C for 30 s. The ΔC_t method was used to calculate the relative expression levels of CamTERFs [53 (link),54 (link)]. ΔC_t = [Gene expression − mean (Actin expression)]/3.

The total RNA was extracted from samples of fungal inoculated leaves using the optimized extraction procedure described by Zhang et al. (2014) (link).
The SYBR Green Premix Ex Taq™ II quantitative PCR system (Takara, Dalian) was used for qPCR analysis. All experiments involving q-PCR were performed on a Q7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The actin gene (GenBank: aK458277.1) was used as the reference gene. The PCR reaction and program were modified according to the manual. The PCR reaction (a total reaction volume of 10 µL) comprised 5 µL 2 × SYBR Green PCR Master Mix, 3 µL of the cDNA product, 1 µL of primer mix, and 1 µL of DNase/RNase-free water. The quantitative PCR thermal cycler program included 95 °C for 10 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 31 s. All primers for q-PCR were synthesized by the same company (AoKe, Yangling) (Table S4).

Total RNA was isolated from three biological replicates of the W931A and W931B samples using RNAiso Plus reagent (TaKaRa, Japan), then treated with RNase-free DNase I (Promega, Madison, WI, USA). Subsequently, 0.5µg RNA per sample was used for first-strand cDNA synthesis using a HiScript II 1st Strand cDNA Synthesis kit (Vazyme, Nanjing, China). qRT−PCR was performed in technical triplicate on a LightCycler 96 (Roche, Switzerland) using the SYBR Green Premix Ex Taq™ II quantitative PCR system (TaKaRa, Japan). The amplification program consisted of a denaturation step at 95°C for 15 min; 40 cycles of 95°C for 10 s and 60°C for 20 s; and 72°C for 30 s. Gene expression was normalized using the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)) using TUBULIN1 as the internal reference gene. All primer sequences are listed in Supplementary Table S1.