4.3. Quantitative RT-PCR (qRT-PCR)

Total RNA was purified from cultured cells using TRIZOL reagent (Thermo Fisher) according to the manufacturer’s protocol. The cDNAs were subsequently prepared from the total RNA using MMLV high-performance reverse transcriptase (Illumina) and oligo(dT) as the primer. The condition for PCR detection was 32 cycles of amplification of the template by denaturing at 94 °C for one minute, primer annealing at 55 °C for 30 s, and product extension at 72 °C for 1 min. Quantitative RT-PCR was performed using GoTag qPCR master mix (Promega, Madison, WI, USA) in a MiniOpticon Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA). The condition for 40 cycles of amplification was template denaturing at 94 °C for one minute, primer annealing at 55 °C for 30 s, and product extension at 72 °C for 45 s. The oligomers used in this study are listed in the supplemental material (Table S1). Student’s t-test was carried out to determine the statistical significance between control and experimental groups.