RNA Extraction and qPCR Analysis of Myocytes

RNA was extracted from myocytes using the HighPure RNA extraction kit (Roche) according to the kit protocol. RNA concentration and purity was determined using the Nanodrop^® ND1000 spectrophotometer [Thermo Scientific and all ratios were within the recommended ranges (A260/280 = 1.8–2.0; A260/230 > 1.7)]. 400 ng total RNA was reverse transcribed to cDNA using the RT² First Strand Kit (Qiagen) according to the manufacturer’s specifications. Quantitative PCR was performed on the cDNA samples using proprietary Quantitect primer assays (Qiagen) (RPLP0, HLA-DPB1) and RT² SYBR Green Mastermix (Qiagen) on the 7900HT Fast Real-Time PCR System (Applied Biosystems). RPLP0 was selected from a panel of 10 reference genes which were screened for their expression stability in myocytes (Nel et al., 2019 (link)). Individual data points were calculated as 2^−ΔCq, where ΔCq = target gene Cq – reference gene Cq (Schmittgen and Livak, 2008 (link)).