Accpower rt premix kit

Total cellular RNA was prepared using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized with 5 µg RNA using an AccPower RT PreMix kit (Bioneer, Daejeon, Korea) at 45℃ for 60 min, and then at 95℃ for 5 min. After cDNA synthesis, primers were added and PCR was performed in a SuperCycler™ apparatus (Kyratec, Queensland, Australia) using 30 cycles of 94℃ for 5 min, 94℃ for 30 sec, 60℃ for 30 sec and 72℃ for 30 sec, followed by 72℃ for 5 min. Preliminary experiments were performed to determine the optimum number of PCR cycles. PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized with SYBR® Green nucleic acid gel stain (Invitrogen). Primer sequences used are shown in Table 1. qRT-PCR was performed using cDNA and specific primers as the template in a 20 µl reaction mixture containing 2×QuantiMix SYBR (PKT, Seoul, Korea). Analysis was carried out using the software supplied with the one-step, real-time PCR System machine (Applied Biosystems, Franklin Lakes, NJ, USA), with each expression calculated relative to mouse β-actin (delta CT) and controls (delta delta CT) using the fluorescence threshold for the amplification reaction and the comparative CT method.