Reverse transcription-quantitative PCR (RT-qPCR)

K562 cells from each experimental group were collected and washed three times with cold PBS, and then total cell mRNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. cDNA was synthesized from 2 µg total RNA using a first-strand cDNA synthesis kit (Thermo Fisher Scientific, Inc.). The PCR amplification protocol was as follows: Denaturation at 94°C for 10 min, followed by 40 cycles of 94°C for 15 sec, 58°C for 30 sec and 72°C for 40 sec. The mRNA expression levels of VDR, Bcl-2, survivin, Bax, p21, p27 and β-actin were detected using ABI 7000 (Thermo Fisher Scientific, Inc.) and Talent qPCR PreMix (SYBR-Green) (Tiangen Biotech Co., Ltd). The relative quantification based on the relative expression of target genes was calculated using the 2^−ΔΔCq method (18). The PCR primers were designed based on the corresponding gene structure, and the sequences are listed in Table I. RT-qPCR was performed in triplicate.

Reverse transcription-quantitative PCR primers.