Quantitative Gene Expression Analysis

Total RNA from the same 8 fish used for sequencing was used to make template cDNA for qPCR analysis. Contaminating DNA in RNA sample was removed by DNAse treatment and cDNA was synthesized using Verso cDNA Synthesis Kit (Thermo Scientific, Hudson, NH). Transcript abundance of mRNA and lncRNA was quantified per manufacturer’s instruction using DyNAmo Flash SYBR Green Master Mix (Thermo Scientific, Hudson, NH) in Bio Rad CFX96™ System (Bio Rad, Hercules, CA). For microRNAs, miScript II RT kit (Qiagen, Valencia, CA, USA) was used to synthesize cDNA, and miScript^R SYBR^R green (Qiagen, Valencia, CA, USA)^{47 (link)} was used to quantify microRNA in Bio Rad CFX96™ System. The endogenous controls used for normalization were B-actin for mRNA and lncRNAs, and U6 for microRNA. None of the endogenous control genes was differentially expressed in this study. Fold changes in gene expression was calculated by using ΔΔCt method as described previously^{35 (link),48 (link)}. Mann-Whitney U test was used to check if the transcript level between atrophying and control muscle was statistically significant (p < 0.05). All 12 transcripts subjected to qPCR validation had Mann Whitney U test p-value < 0.05.