2.8 Real-time polymerase chain reaction (PCR)
Total RNA (1 μg) extracted from atrial tissue using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as previously described (Aonuma et al., 2020; Feng et al., 2020; Xu et al., 2012; Xu et al., 2010). RT-PCR was performed on the ABI Prism 7500 FAST sequence detection system (Applied Biosystems, Foster City, CA, United States) using the PrimeTime Gene Expression Master Mix (Integrated DNA Technologies). The following primers were used: Nppb (Mm.PT.588584045.g), Col1a1 (Mm.PT.587562513), Tgfb1 (Mm.PT.5811254750), Ryr2 (Mm.PT.58.45974879), Camkiia (Mm.PT.58.30362101), and Cacna1c (Mm.PT.58.16361922). Gene expression was normalized to that of the housekeeping gene, 18S rRNA (4319413E; Thermo Fisher Scientific).