Muscle Gene Expression Analysis Pipeline

Muscle RNA extraction, reverse transcription, and qPCR were performed following the procedures established for our laboratory [35 (link)]. RNA was extracted using RNAiso Plus (Takara Bio, Kusatsu, Japan), treated with DNase I (Beyotime), and analyzed for purity and quantity via agarose gel electrophoresis and spectrophotometry (A_260/280 ratio), respectively. Reverse transcription was performed using the PrimeScript™ RT Reagent Kit (Takara Bio). The reaction system for qPCR contained 1 µL complementary DNA, 0.2 µL ROX, 5 µL SYBR Premix Ex Taq, 0.4 µL primers, and 3.4 µL nuclease-free water (Vazyme, Nanjing, China). The reaction was run in a QuanStudio 5Real-Time System (Life Technologies, Carlsbad, CA, USA) according to manufacturer protocol. The oligonucleotide primers used are presented in Table S4. The 2^−ΔΔCT method was used to calculate the changes in target gene expression relative to β-actin levels.