To perform real-time qPCR utilizing a LightCycler 480 (Roche Diagnostics K.K.), TaqMan® Gene Expression Assays (Life Technologies Japan Ltd.) were used as a probe/primer set specified for the KCNJ6 gene (Assay ID: Hs01040524_m1) and a probe/primer set for the ACTB gene, a house-keeping gene, encoding β-actin (Assay ID: Hs99999903_m1). PCR was performed in a final volume of 20 µl containing 2×LightCycler® 480 Probes Master, 1 µl TaqMan® Gene Expression Assay, 1 µl cDNA as the template, and up to 20 µl H2O equipped with 2×LightCycler® 480 Probes Master. The PCR program was the following: 95°C for 10 min, 45 cycles of 95°C for 10 s and 60°C for 30 s, followed by 95°C for 10 s, 50°C for 30 s, 50–70°C (continuously) at a rate 0.06°C/s, with a final cooling at 50°C for 30 s. The expression level of the KCNJ6 gene was normalized to that of the ACTB gene for each sample, and relative KCNJ6 mRNA expression levels between all samples were compared by setting the lowest expression level among all SMRI samples as 1. Experiments were performed in duplicate (separate experiments) for each sample, and averaged values were calculated for normalized expression levels.
