RT-qPCR Analysis of Gene Expression

RNA was reverse transcribed into cDNA using Takara PrimeScript RT master mix (RR036A; Takara Biotechnology Co., Ltd., Dalian China). RT-qPCR was performed using an ABI StepOne Plus Real-Time PCR system (Thermo Fisher Scientific, Inc.) and SYBR Premix Ex Taq II master mix (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. The reaction system (10 µl) consisted of cDNA (1 µl), forward primers (10 µM; 0.2 µl), reverse primers (10 µM; 0.2 µl), ROX reference dye (0.2 µl), RNase-free water (3.4 µl), and SYBR-Green mixture (5 µl). The thermocycling conditions were as follows: Initial denaturation, 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Rat actin was used as a housekeeping gene. The relative expression of genes was calculated using the 2^−∆∆Ct method [5 (link)].