Real time pcr easy sybr green 1

Reverse transcribed cDNA products were used as templates of quantitative Real-Time PCR (qRT-PCR). The reaction was carried out on a CFX Opus Real-time PCR system using the Real-Time PCR Easy ™ -SYBR Green I (Foregene, Ltd., Chengdu, China) following the manufacturer’s instructions. NCBI-BLAST Primer designed the primers for 35 SmHSF genes. S. miltiorrhiza Actin was used as an endogenous control for the normalization of expression levels of genes (Jiang et al., 2020 (link)). The relative expression levels were calculated using the 2^−ΔΔ Ct method. Data were analyzed using one-way ANOVA in GraphPad Prism 9 software (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). To ensure reproducibility and dependability, three biological replications and three technical replications were implemented for each sample. The primers for the SmHSFs used for qRT-PCR analyses are listed in Table S2.

The validation was performed with the same RNA samples used for RNA-seq analysis. The three biological replicates of the four varieties of S. miltiorrhiza (SC, YN, SX, and YN) at two accumulation stages were analyzed. cDNA synthesis was done by using 400 ng of the total RNA samples with RT Easy™II Kit (Foregene CO., LTD., Chengdu, China). For quantitative PCR, reverse transcribed cDNA products were used as templates. Differentially expressed genes related to tanshinones biosynthesis and candidate transcription factors related to tanshinones accumulation were selected for validation. The primers employed in the qRT-PCR experiments were designed according to the assembled sequences. Primer sequences were reported in Table S1. Actin was used as an endogenous control for the normalization of expression levels of genes (Jiang et al., 2020 (link)). The reaction was carried out on a CFX Opus Real-Time PCR System using the Real-Time PCR Easy™-SYBR Green I (Foregene CO., LTD., Chengdu, China) with a total reaction volume of 20 µL, 0.4 μM of the primer, and 80 ng of cDNA. Relative gene expression levels were calculated using the 2^−ΔΔCt method (Yuan et al., 2006 (link)). To ensure reproducibility and reliability, three biological replications and three technical replications were implemented for each sample.