RNA extraction and reverse transcription-quantitative (RT-q) PCR

After treatment, total RNA was extracted from each group of cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and the RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). Next, mRNA was reverse-transcribed into cDNA using a reverse transcription system (Toyobo Life Science) according to the manufacturer's protocol. Then, cDNA was used as a template to perform qPCR (BeyoFast SYBR Green qPCR Mix; Beyotime Institute of Biotechnology) under the following thermal cycler conditions: 95˚C for 5 sec, followed by 45 cycles, including denaturation at 95˚C for 30 sec in an ABI 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.); further it was annealed at 60˚C for 30 sec and extended at 72˚C for 30 sec. GAPDH was used as an internal control, and the relative expression level of mRNA was calculated using the 2^-ΔΔCq method (21). Primers were designed and synthesized by BGI Biological Co. and the sequences are listed in Table I.

Primers used for quantitative PCR.