Measurement of target gene in myocardium by RT-qPCR

Total RNA was extracted from myocardial tissue (Ultrapure RNA extraction Kit (#CW0581, ComWin Biotech CO., LTD., Beijing, China)). Absorbance was measured using an ultraviolet spectrophotometer and purification was evaluated by the ratio of absorbance at 260 and 280 nm (OD260/OD280). Ratio ranges between 1.8 and 2.0 indicate that the RNA is highly purified and suitable for real-time quantitative polymerase chain reaction (RT-qPCR). Reverse transcription was carried out using a cDNA First Chain Synthesis Kit HiFi-MMLV (#CW0744, ComWin Biotech CO., LTD., Beijing, China) and amplified using UltraSYBR Mixture (with Rox) (#CW0956, ComWin Biotech CO., LTD., Beijing, China), according to the manufacturer’s instructions. The target gene and housekeeping gene were all purchased from Guangzhou FulenGen CO., LTD., Guangzhou, China: TGF-β1 (Cat#RQP050181), CTGF (Cat#RQP050397), GAPDH (Cat#RQP049537). Relative quantitative analysis of mRNA was carried out using the 2^−ΔΔCt method.