Reverse transcription 3

Isolated soleus muscle segments were disrupted in TissueLyser (QIAGEN). RNA isolation was performed with the Mini kit (QIAGEN) in a QIAcube robotic workstation for automated purification of RNA (QIAGEN). PCR was performed to assess the expression of specific RNAs. The reaction was executed with reverse transcription III (Invitrogen) and SUPERase (Ambion) for deactivation of RNase and DNase. The standard primer sets for quantitative PCR analyses for α1 and α2 isoforms of the Na,K-ATPase were obtained from Applied Biosystems. Quantitative PCR was performed on an MX3000P (Agilent Technologies) using TaqMan probe (FAM) technology. Gene expression was normalized to GAPDH and transferrin receptor (Ct value) levels and presented by a ΔCt value. Comparison of gene expression was derived by subtracting control ΔCt (an averaged ΔCt for the muscles which were not exposed to HS) from sample ΔCt, producing ΔΔCt. Relative gene expression was calculated as 1/(2^ΔΔCt), thereby standardizing to control muscle.

Total RNA was isolated from cells and tissue using TRIzol reagent. Primer sequences used for RT-PCR and ChIP-PCR, as well as product numbers for real-time TaqMan primers (Applied Biosystems) are listed in Table S1. For real-time PCR, gene expression was determined using the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). mRNA was primed with random hexamers and reverse transcribed with Reverse Transcription III (Invitrogen). RT-PCR was performed on a GeneAmp PCR System 3700 (Applied Biosystems) under the default parameters for 35 cycles. For real-time PCR, samples were prepared using TaqMan Gene Expression Master Mix (Applied Biosystems) and TaqMan probes (Table S1) on a qPCR system (Mx3000P; Agilent Technologies), using the default parameters for 35 cycles.