Tiangen reagent

As previously described (5 (link)), TIANGEN reagent (TIANGEN Biotech, Beijing, China) was used to extract total RNA from homogenized tissue or cultured cells, following the manufacturer's instructions. The reaction solution consisted of 2.0 μl of diluted RT-PCR product, a 0.2 μm concentration of each paired primer, and power SYBR Green PCR master mix (Toyobo, Osaka, Japan). The Kv4.2 primer sequence was 5′-TGTCAGGAAGTCATAGAGGCAGCGTG-3′ (forward) and 5′-GGGGTGGTTACTGGAGGTGTTGGAAT-3′ (reverse). The sequence of housekeeping gene cyclophilin D, used as a control to exclude sampling errors, was 5′-GGACGTCTGTCTTCGAGTCC-3′ (forward) and 5′-AACAGACCGTGGAGATTTGG-3′ (reverse). The annealing temperature was set at 58 °C for Kv4.2 and 61 °C for cyclophilin D, with 38 amplification cycles for each product. The absolute mRNA levels in each sample were calculated according to a standard curve set up using serial dilutions of known amounts of specific templates against corresponding cycle threshold (Ct) values. The normalized ratio of Kv4.2 to cyclophilin D in each group was presented. The specificity of the primers was verified by both gel electrophoresis and sequencing of the PCR products.

Total RNAs from S. miltiorrhiza Bunge roots were extracted using the TIANGEN reagent according to the manufacturer’s instructions (TIANGEN, China). The DNase-treated RNA was reverse transcribed using SuperScript reverse transcriptase (TaKaRa, China) according to the manufacturer’s instructions. Real-time quantitative PCR was performed on an optical 96-well plate with an ABI PRISM 7500 real-time PCR system (Applied Biosystems) using SYBR Premix ExTaq (TaKaRa, China). The PCR method was programmed as follows: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, and 60°C for 30 s. The SmActin gene was used as the endogenous control. The quantitative RT-PCR primer is shown in supplementary S2 Table. These experiments were repeated three times.