Transscript uni all in one first strand cdna synthesis supermix for qpcr kit

Total RNA was extracted using the Total RNA Isolation Reagent Kit (Magen, China) according to the manufacturer’s protocol. RNA was reverse-transcribed into cDNA using the TransScript® Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit (TransGen Biotech, China) and quantitative PCR was performed using SYBR Premix (Vazyme Biotech Co., Ltd.). The specific primer sequences for each mRNA were listed in Additional file 1: Table S1. All the experiments were performed on QuantStudio 1.

Total RNA of plant samples was extracted using TIANGEN’s RNAiso Plus reagent (Tiangen Company, Beijing, China). TransScript^® Uni All-in-one First-strand cDNA Synthesis SuperMix for qPCR kit (TransGen Biotech Co., Ltd., Beijing, China) was used for both reverse transcription and qRT-PCR experiments using GmEF1A as an internal reference gene. Each qPCR experiment was repeated with three biological replicates, with the relative gene expression calculated according to the method of 2^−ΔΔCt. The primers used in the qPCR experiments were synthesized by Shanghai Biotech Bioengineering Co., Ltd. (Shanghai, China; Table 4).

Nine DEGs highly correlated with flavonoid and alkaloids synthesis were verified via the qRT-PCR method. Next, 1 µg of each qualified RNA sample was used for the reverse-transcription reaction. The RNA samples were reverse-transcribed to first-strand cDNA with a TransScript^® Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit (TransGen Biotech., Beijing, China) according to the manufacturer’s instructions. The primers utilized in this study are comprehensively listed in Table S1. The qRT-PCR method was conducted with a Magic SYBR Mixture qPCR kit (Cwbio Bio., Beijing, China). The reaction system was as follows: 1 µL of cDNA, 10 µL of 2 × PerfectStart Green qPCR SuperMix, 0.4 µL of forward primer (10 µM), 0.4 µL of reverse primer (10 µM), and 8.2 µL of nuclease-free water. The qRT-PCR was performed on an Archimed-X4 fluorescence quantitative PCR instrument (ROCGENE, Beijing, China). Relative transcript levels were determined using the 2−ΔΔCt method, with β-Actin gene serving as the reference gene. The experiments were replicated both biologically and technically, with a total of three replicates performed for each sample. The data were graphed using OriginPro 9.1 software (OriginLab Corp., Northampton, MA, USA).