Lightcycler 480 2 quantitative pcr system

Total RNA from transfected cells was collected, placed in Trizol reagent (Invitrogen), homogenized for 20 seconds and incubated on ice for 5 min. Then, chloroform, isopropanol and absolute ethanol were sequentially added and centrifuged at 12000 r.p.m. after each addition for ten minutes at 4 °C. The precipitate was obtained and dried at room temperature for 10 min and then added to the reverse transcription reaction system. Quantitative real-time PCR (qRT-PCR) was performed to detect relative mRNAs using a PrimeScript™ RT Reagent Kit (Takara, Japan). For the miRNAs, cDNA was synthesized using a MiR-X™ miRNA First-Strand Synthesis Kit (Takara), and qRT-PCR was performed using a TB Green™ Kit (Takara). qRT-PCR was performed in triplicate using a LightCycler 480 II quantitative PCR system (Roche, Switzerland). A comparative Ct (2^−ΔΔCT) method was used for analysis, and U6 and GAPDH served as internal reference genes. The primers for qRT-PCR are shown in Supplemental Table S1.

Total RNA (500 ng) extracted from PASMCs by trizol reagent according to the protocol of the manufacturer (life technologies). The cDNA was generated by using PrimeScript RT reagent Kit (TAKARA) with oligo-dT and random primer. Real-time PCR was performed on a LightCycler 480 II quantitative PCR system (Roche) using SYBR Green. Primers used in the amplification reaction were in Table S3.