Quantitative Analysis of Na+,K+-ATPase Subunits

Total RNA was isolated using RNeasy Plus Mini Kit (Qiagen) or E.Z.N.A. HP Total RNA Kit (Omega Bio-tek) and reverse transcribed to cDNA with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative real-time polymerase chain reaction (qPCR) was performed on 7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific) using TaqMan Universal MasterMix and TaqMan gene expression assays for human α1-subunit of Na⁺,K⁺-ATPase (No. Hs00167556_m1) and human α3-subunit of Na⁺,K⁺-ATPase (No. Hs00958036_m1). Expression of target gene was normalized to expression of cyclophilin (PPIA, No. Hs99999904_m1), which was used as an endogenous control. Efficiency of PCR was estimated with LinRegPCR software (2018.0) (Ramakers et al. 2003 (link); Ruijter et al. 2009 (link); Tuomi et al. 2010 (link)).