RNA Extraction and Real-time PCR Analysis

An RNAiso Plus Purification Kit (TaKaRa Biotechnology Co., Ltd, 9108) was used to extract total RNA from the cardiomyocytes. The RNA concentration was evaluated based on the optical density of the sample at 260 nm, and the RNA integrity was assessed through 2% agarose gel electrophoresis. The RNA was reverse-transcribed into cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa Biotechnology Co., Ltd, RR047A) [71 (link), 72 (link)]. Real-time PCR was performed on a LightCycler machine (Roche) with a commercial SYBR Green reaction reagent (TaKaRa Biotechnology Co., Ltd, RR820A). GAPDH was used as an internal control. The cDNA was denatured for 30 s at 95 °C, followed by 40 cycles of 5 s at 95°C [73 (link)].