Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the RNA concentration was determined using a UV spectrophotometer. RT of total RNA to cDNA was performed using an AccuPower RocketScript^™ RT PreMix kit (Bioneer Corporation, Daejeon, Korea), following the manufacturer's protocol. The thermal cycle profile for RT was set for primer annealing at 37°C for 10 min, cDNA synthesis at 50°C for 60 min and heat inactivation at 95°C for 5 min. qPCR was performed using an AccuPower 2× GreenStar^™ qPCR Master Mix kit (Bioneer Corporation) in a Bio-Rad iQ5 optical module (Bio-Rad Laboratories, Inc., Hercules, CA, USA). qPCR was performed under the following conditions: 95°C for 10 min, and 40 cycles of denaturation at 95°C for 10 sec and annealing at 58°C for 20 sec. Primer sequences were: GRP78 forward, 5′-TGACTATGAAGAATCCCAAGA-3′ and reverse, 5′-TATCAACATCCAGTTCCACC-3′); GAPDH forward, 5′-GTTCAACGGCACAGTCAAGG-3′ and reverse, 5′-CACCAGTGGATGCAGGGAT-3′. 2^−ΔΔCq was calculated for every sample, and the mRNA expression levels were determined using the 2^−ΔΔCq method (14) and normalized to GAPDH.