Quantitative RT-PCR Analysis of Cardiac Markers

mRNAs were extracted from ventricular tissues or cell lysates using RNeasy Mini kits (Qiagen, Cat. No. 74104, Hilden, Germany). Reverse transcription was performed using an iScript cDNA synthesis kit (Bio Rad, Cat. No. 1708891, Hercules, CA, USA). PCR reactions were performed with an ABI Step One Real-Time PCR System (Applied Biosystems, Quant Studio3, Foster, CA, USA) using Fast SYBR Green Real-time PCR Mixture (Applied Biosystems, Cat. No. 4385612, Foster, CA, USA) [52 (link)]. Primers for CTGF (Takara Bio Inc. Cat. No. MA028643), TGF-β (Takara Bio Inc. Cat. No. MA122075), collagen I (Takara Bio Inc. Cat. No. MA128559), collagen III (Takara Bio Inc. Cat. No. MA126692), OPN (Takara Bio Inc. Cat. No. MA114392) (Takara Bio Inc. Cat. No. HA201672), MCP-1 (Takara Bio Inc. Cat. No. MA066003), HGF (Takara Bio Inc. Cat. No. MA105799), and 18S ribosomal RNA (18S) (Takara Bio Inc. Cat. No. MA050364) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Takara Bio Inc. Cat. No. HA067812) are available commercially. The mouse heart samples were normalized to values for 18s mRNA expression (ΔΔCT method). The human cardiomyocyte culture samples were normalized to values for GAPDH mRNA expression (ΔΔCT method).