Gene Expression Analysis Protocol

Total RNA was extracted using the HiGene Total RNA Prep Kit (BIOFACT, Daejeon, Korea) and reverse-transcribed to cDNA using the Reverse-Transcription Premix kit (Elpis Biotech, Daejeon, Korea). Real-time qPCR was conducted using the CFX96 Touch Real-Time PCR detection system (Bio-Rad Laboratories) and TOPreal qPCR 2X PreMIX (SYBR Green with low ROX) (Enzynomics, Daejeon, Korea). Amplification reactions were performed at 95°C for 15 min, followed by 40 cycles of 95°C for 20 sec, 59°C for 20 sec, and 72°C for 30 sec. SYBR green fluorescent signals were assessed using the iQTM5 optical system software (Bio-Rad Laboratories). The relative expression of target genes was compared with that of the control group after normalizing to C_t values of GAPDH. Except for the P53 primer sequence, the target gene primer sequences were obtained from OriGene Technologies (Rockville, MD, USA). Bassiony et al. [20 (link)] provided the P53 primer sequence. Supplementary Table S1 shows the target gene primer sequences.