RNA isolation and quantitative RT–PCR

Total RNA was extracted from liver tissues using an easy-spin^TM total RNA extraction kit (Intron Biotechnology, Korea) according to the manufacturer’s protocol. Total RNA (4 µg) was used for cDNA synthesis using random hexamers, oligo dT primers, and reverse transcription master mix (EBT-1511, EBT-1512; ELPIS Biotech, Korea). Quantitative real-time PCR was performed using the cDNAs and each gene-specific oligonucleotide primer in the presence of TOPreal qPCR premix (RT500M; Enzynomics, Korea). The following real-time PCR conditions were used: an initial denaturation step at 95 °C for 15 min, followed by 50 cycles of denaturation at 95 °C for 10 sec, annealing at 58 °C for 15 sec, and elongation at 72 °C for 20 sec. Each PCR product was evaluated by melting curve analysis for quality control. The qRT-PCR data were collected using LightCycler 480 software 1.5.0 (Roche). Supplementary Table ¹ shows the sequences of the gene-specific primers used for qRT-PCR.