RNA Extraction and Gene Expression Analysis

In accordance with the manufacturers’ protocols, Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and RNeasy mini kits (Qiagen, Valencia, CA, USA) were used for the extraction of total RNA. Tri-reagent was used to homogenize muscle and the subsequent extraction was performed in phenol/chloroform. Seventy percent ethanol was added to the aqueous phase and the sample was then applied to a Qiagen minispin column, and the protocol from the manufacturer was followed [20 (link)]. RNA was eluted from the column using RNase-free water and then quantified (Thermo Fisher Scientific, Waltham, MA, USA). RNA quality was assessed using 1% agarose gel, and total RNA was reverse-transcribed (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. An aliquot of the reverse-transcribed reaction mix was used for qPCR and TaqMan gene expression assays: CAT (cationic amino acid transporter)-1 (NM_013111.2), SNAT (sodium-coupled neutral amino acid transporter)-2 (NM_181090.2), LAT (large neutral amino acid transporter)-2 (NM_019283.1), PAT (proton-coupled amino acid transport)-1 (NM_130415.1), PAT-2 (NM_139339.1), and PAT4 (NM_001108127.1) (Applied Biosystems, Foster City, CA, USA). The endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH, NM_017008.3) was used in the expression of target genes by the 2^−ΔΔCt method.