RNA Extraction and qRT-PCR Analysis

H9c2, NMCMs, and hiPSC-CMs were collected into QIAzol Lysis Reagent or Trizol Reagent (Invitrogen) and total RNA was extracted using Direct-zol RNA Miniprep and Microprep (Zymo Research, CA, USA), respectively. Heart samples were first homogenized in QIAzol Lysis Reagent using the Qiagen TissueLyser LT Bead Mill (QIAGEN, Venlo, Netherlands) and total RNA was thereafter extracted using Direct-zol RNA Miniprep. RNA was reverse-transcribed to cDNA (PrimeScript RT reagent kit, Takara Clontech) and quantitative real-time polymerase chain reaction (RT-PCR) was performed with SYBR Green protocols (Kapa Biosystems, MA, USA) and a real-time PCR detection system (Applied Biosystems 7300 Real-Time PCR system) (Rinne et al, 2017 (link); Kadiri et al, 2021 (link)). Target gene expression was normalized to a housekeeping gene (ribosomal protein S18; RPS18, glyceraldehyde-3-phosphate dehydrogenase; GAPDH or β-actin; ACTB) using the comparative ΔCt method and results are presented as relative transcript levels (2^-ΔΔCt). Primer sequences are presented in Appendix Tables S4–S6.