Chamq universal sybr qpcr master kit

The aboveground tissue was quickly frozen with liquid nitrogen and ground into a powder with a mortar. Total RNA was extracted using TRIzol reagent. First-strand cDNA was synthesized from 3 μg of total RNA in a 20 μL volume using an Oligo(dT)₁₈ primer with TransScript II One-Step gDNA Removal (TransGen Biotech. Cat. No. AT311-03) and cDNA Synthesis SuperMix time PCR was performed on Biometra Tone 96 G according to a two-step method using the ChamQ Universal SYBR qPCR Master kit (Vazyme. Cat. No. Q711-02). Briefly, each 10 μL qRT–PCR contained 4.6 μL of 20-fold diluted cDNA, 0.2 μL of each primer and 5 μL ChamQ Universal SYBR Qpcr Master Mix. The reaction procedure was predenaturated at 95 °C for 30s, followed by a 40-cycle program (95°C 10 s and 60°C 30s). GmActin (Glyma.18G290800) was used as an internal control to calculate the transcript levels of selected genes, which were quantified by the cycle threshold 2^-ΔCt method. △Ct=Ct (selected gene)-Ct (GmActin). Each set of experiments was performed with at least three biological replicates and two technical replicates. Related genes and primer lists are presented in Table S1.

RNA extraction was carried out using the Tatal RNA Kit I R6834 (Omega Bio-tek, Norcross, GA, USA) as per the manufacturer’s instructions. cDNA synthesis was performed using the HiScript II Q Select RT SuperMix (Vazyme, Nanjing, China) and the RT-PCR was carried out with the ChamQ Universal SYBR qPCR Master Kit (Vazyme) as per the manufacturer’s instructions. The fold change of gene expression was normalized with 18S ribosomal RNA and relative fold change was calculated using the 2^−ΔΔCT method. All primer sequences used for RT-PCR are listed in Table 1.