Sybr green master mix no rox

Total RNA from the different tissues and leaf axils was extracted with an RNAprep Pure Plant Kit (TIANGEN, Beijing, China) according to the kit protocol, and DNA contamination was removed with RNase-free DNase I. First-strand cDNA was synthesized with a Hifair^®Ⅱ 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yeasen, Shanghai, China) according to the kit protocol. Gene-specific primers for qRT-PCR were designed with Primer 6.0 (Supplemental Table S2). The LilyActin primer was used as an internal control [66 (link)], and SYBR^® Green Master Mix (No Rox) (Yeasen, Shanghai, China) was used in the reaction mixture according to the manufacturer’s instructions. qRT-PCR was conducted using the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA), with an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s and extension at 72 °C for 1 min. A melting curve analysis was performed for each primer pair to confirm its specificity. The 2^−ΔΔCt method was used to calculate the relative expression levels of the different genes [67 (link)]. Three biological and three technical replicates were used to reduce error.

To examine the levels of cellular inflammatory factor mRNA, qPCR was performed as previously described [35 (link)]. PvTRAgs were co-incubated with HSFs after 48 h total RNA had been isolated from HSFs in accordance with the protocols of the manufacturer (Yeasen Biotechnology, Shanghai, China). The extracted total RNA was further processed to remove genomic DNA and then reverse transcribed into complementary DNA (cDNA) using 4 × HiFiScript RTMaster Mix, following the instructions for the RNA Reverse Transcription Kit (Yeasen Biotechnology, Shanghai, China). RT-PCR was performed in triplicate with three independent samples for each experimental group in a LightCycler480 II apparatus (Roche, USA) with SYBR Green Master Mix (No Rox) (Yeasen Biotechnology, Shanghai, China). The amplification program followed a two-step method. Thermal cycling conditions were as follows: pre-denaturation at 95 ℃ for 5 min, followed by 40 PCR cycles at 95 ℃ for 10 s and 60 ℃ for 30 s. The ratio of each target gene was determined using GAPDH as an internal control. The relative expression levels of genes were calculated from the quantification cycle (Cq) value and standardized by the 2^−ΔΔCq method. Primers for amplified genes (IL-1β, IL-6, and TNF-α) are presented in Additional file 2: Table S2.