2.7. Validation of miRNA Expression Using Quantitative Real-Time PCR (RT-PCR)

In order to validate initial miRNA sequence results, the 10 most significant up- or downregulated miRNAs were selected for further RT-PCR tests as reported previously [14]. Total RNA was isolated using TRIzol reagent and the quality and quantity of RNA was measured using a NanoDrop 2000 spectrophotometer. Each reverse transcription reaction mixture contained 10 mL of SYBR Green Master Mix, 0.5 mL of miR-RT primers F (10 mM), 0.5 mL of miR-RT primers R (10 mM), and RNase-free H₂O. The RT-PCR reactions for the selected 10 miRNAs were performed using the ViiA 7 Real-Time PCR System (ABI, USA) under the following conditions: 95°C for 1 min, followed by 40 PCR cycles (95°C for 10 s and then 60°C for 20 s). miRNA expression was normalized to the endogenous reference gene GAPDH. Each sample was analyzed in triplicate. Specific primers were produced by BIOTNT Company (Shanghai, China). Relative quantification was achieved by the comparative 2^−ΔΔct method.