Primescript reverse transcription master mix

Total RNA was extracted using the standard TRIzol method, and yields/purity were measured using the Nanodrop 2000 (Thermo Fisher Scientific, USA). Total RNA was reverse transcribed into cDNA using PrimeScript^™ Reverse Transcription Master Mix (code No. RR036A; Takara) and subsequently used for PCR amplification of NSUN6 and NM23-H1 (and GAPDH as internal control) using an ABI StepOne Plus System (Applied Biosystems, NJ, USA). There were 40 cycles of 95°C for 5 min, 95°C for 20 s, and 59°C for 15 s. Relative expression was calculated using the 2^−ΔΔCt method. The primer sequences used for amplification were NSUN6 sense, 5′-ATC TGC GTC CGT TTC ACC-3′, and antisense, 5′-GCT TCC ACC ACA CCT CAT C-3′; NM23-H1 sense, 5′-GCA GCC GGA GTT CAA ACC TA-3′, and antisense, 5′-TGC ACA CCA GGC TGA CTT AG-3′; GAPDH sense, 5′-TAT GAT GAT ATC AAG AGG GTA GT-3′, and antisense, 5′-TGT ATC CAA ACT CAT TGT CAT AC-3′.

Total RNA was extracted from myocardium using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and reverse-transcribed into complementary DNA (cDNA) with a PrimeScript Reverse Transcription Master Mix (TaKaRa, Shiga, Japan), according to the protocol per manufacturer’s instruction. Quantitative real-time PCR (qPCR) was performed with TB Green premix Ex Taq II (TaKaRa) on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). In order to analyze relative gene expression with real-time PCR, we selected two reference genes: β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The intra- and inter-assay variation was tested. The real-time PCR efficiency of the two reference genes was similar, and expression of the two reference genes was constant. We selected GAPDH as reference gene and expression of the target gene was normalized to GAPDH. The primers listed in Table 1. Relative expression levels were calculated using the cycle threshold (2^−ΔΔCt) method.