RNA extraction and real‐time quantitative PCR

Total RNA was isolated using Tri‐reagent (Molecular Research Center, Cincinnati, OH) and RNeasy mini kit (Qiagen, Valencia, CA) according to manufacturers’ protocol, as previously reported (Lang et al. 2012). Briefly, gastrocnemius was homogenized in Tri‐reagent followed by chloroform extraction. An equivalent volume of 70% ethanol was added to the aqueous portion and loaded to the Qiagen mini‐spin column. The protocol of the Qiagen mini‐kit protocol was then performed including the on‐column DNase I treatment. RNA was eluted from the column with RNase‐free water and quantified (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA). RNA quality was evaluated on 1% agarose gel. Total RNA was reverse transcribed using superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). Real‐time quantitative PCR was performed using 25 ng of cDNA in StepOnePlus system using TaqMan gene expression assay (Applied Biosystems, Foster, CA) for: atrogin‐1 (NM_026346.2), muscle RING‐finger 1 (MuRF1; {"type":"entrez-nucleotide","attrs":{"text":"NM_001039048.2","term_id":"124244069"}}NM_001039048.2), interleukin (IL)‐6 (Mm00446190_m1), TNF‐α (Mm00443258_m1), ribosomal protein L32 (rpL32; Mm02528467_g1), and DNA‐directed RNA polymerase II polypeptide A (Polr2a; Mm00839493_m1). The comparative quantitation method 2^−∆∆Ct was used to present gene expression normalized to the endogenous control, rpL32 for the acute sepsis study and Polr2a for chronic sepsis studies. In chronic sepsis, rpL32 was not used as a control due to a significant variation in the mRNA content between groups, but Polr2a did not differ between groups.