Quantitative Gene Expression Analysis

Total RNA from DSA and PRA was extracted using AG RNAex Pro Reagent (Accurate Biotechnology (Hunan), Co., Ltd., China) according to the manufacturer’s instructions. The purity of the total RNA was verified using a NanoDrop^® ND-2000 spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA). The removal of gDNA in the total RNA and reverse transcription were accomplished by using Evo M-MLV RT Kit with gDNA clean for qPCR (Accurate Biotechnology (Hunan), Co., Ltd., China). Real-time PCR was conducted with an ABI 7900 PCR system (ABI Biotechnology, Eldersburg, MD, USA). The total reaction system (10 μL) included 2 μL cDNA (5 ng/μL), 5 μL SYBR Green Pro Taq HS Premix (Accurate Biotechnology (Hunan), Co., Ltd., China), 0.2 μL of each primer (10 μM) and 2.6 μL of RNase-free water. The PCR protocol was: incubation for 10 min at 95 °C, followed by 40 cycles of denaturation for 15 s at 95 °C, and annealing and extension for 60 s at 56 °C to 64 °C. All the samples were measured in duplicate. The primers were designed using the Oligo 6.0 software (Table 1). Relative expression of target genes was calculated by the 2^−ΔΔCt method [12 (link)].