Quantitative RT-PCR Analysis of Kv4.2 Expression

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.