Detection of p53, p21 and Ki67 mRNA expression in transplanted tumor tissues by reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from the tumor tissues using TRIzol^® reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Pectrophotometric quantification was utilized to determine RNA purity using an OD ratio of 260 nm/280 nm and a BeekmanDU 800 UV spectrophotometer. A total of 1 mg total RNA was reverse transcribed into first-strand cDNA using the Revert Aid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using the Taq-Man™ Gene Expression assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) on an Mx3000P real-time PCR system (Agilent Technologies, Inc.) according to the manufacturer's instructions. The primer pairs used in the present study were designed and synthesized by Sangon Biotech Co., Ltd. (Table I). The following thermocycling conditions were used: Initial denaturation at 95˚C for 5 min; 35 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 30 sec, elongation at 60˚C for 30 sec and a final extension step at 60˚C for 40 sec. mRNA levels were normalized to the internal reference gene GAPDH. ^∆∆CT values of each group were calculated separately=[(CT experimental target gene - CT internal reference target gene)-(CT control target gene-CT internal reference control gene)]. mRNA relative expression was calculated using 2^-∆∆Cq values (28).

Primer sequences used for reverse transcription-quantitative PCR.