Rt pcr reagent kit

Total RNA was extracted from the cells in each group using TRIzol^® (Invitrogen). RNA was reverse transcribed to cDNA using the PrimeScript 1^st Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's instructions. RT-qPCR was performed using an RT-PCR reagent kit (Takara Biotechnology Co., Ltd.), according to the manufacturer's instructions. β-actin was used as an internal control. The primers for DOR, MDR1 and β-actin were synthesized by Invitrogen, and the primer sequences are presented in Table I. The qPCR reaction contained a total volume of 50 µl (2 µl template, 2 µl primer 1 (10 µM), 2 µl primer 2 (10 µM), 25 µl PCR MasterMix, 19 µl ddH₂O). An SYBR^® Green-based RT-qPCR assay (Beyotime Institute of Biotechnology, Shanghai, China)was used to determine the mRNA expression levels using an ABI PRISM 7900HT Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the cycling conditions were as follows: 94°C for 2 min, followed by 30 cycles of dena-turation at 94°C for 30 sec, annealing at 64°C for 30 sec, and extension at 72°C for 30 sec. β-actin was used as an internal control to normalize gene expression levels. The PCR products were subsequently subjected to 1.0% agarose gel electrophoresis, and the results were scanned and analyzed using a gel documentation system (Syngene, Cambridge, UK).