Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNAs were extracted from cells using a Trizol kit (Invitrogen, Carlsbad, CA, USA) and reversely transcribed into complementary DNA (cDNA) according to the instructions of TaqMan MicroRNA Assays Reverse Transcription Primer (4427975; Applied Bio-systems, Foster City, CA, USA). The conditions of reverse transcription reaction were 37°C for 30 min and 85°C for 5 s. Then 5 μL of cDNA obtained above was taken as template for quantitive polymerase chain reaction (PCR) amplification using a QuantiTect SYBR Green RT-PCR kit. The reaction conditions were comprised of pre-denaturation at 95°C for 5 min, 45 cycles of denaturation at 95°C for 20 s, annealing at 60°C for 1 min, and extension at 72°C for 30 s. The expression of each gene was analyzed. Real-time quantitative PCR results were analyzed using the 2-Dct theory. U6 was served as an internal reference for miRNA, and β-actin was served as an internal reference for the other genes. The fold changes were calculated by means of relative quantification (2-ΔΔCt method) (Livak and Schmittgen, 2001). The primers are depicted in Table 1.