Total RNA was extracted from the longissimus dorsi muscles, C2C12 myoblasts, or sheep skeletal muscle SCs with TRIzol reagent (Invitrogen, USA), according to manufacturer’s instructions. For mRNA quantification, 1 μL of total RNA (1000 ng/μL) was reverse transcribed into cDNA using the PrimeScript RT reagent Kit (Perfect Real Time; Takara, Japan) for quantitative real-time PCR (qPCR). GAPDH and ACTB genes were used as internal normalization controls. qPCR was carried out in an Applied Biosystems 7500 Real-Time Detection system using the SYBR premix Ex Taq qPCR Kit (Takara, Japan).
For miRNA quantification, a miRNA stem-loop primer and a pair of primers were designed for miRNA reverse transcription and qPCR, respectively. The U6 gene was used as the internal normalization control. MiRNA stem-loop was reverse transcribed with the PrimeScript RT reagent Kit and quantified by qPCR using the SYBR premix Ex Taq qPCR Kit, according to manufacturer’s protocols. Relative gene expression was determined by the 2−ΔΔCt method50 (link)51 (link). The primers used for qPCR are listed inSupplementary Tables S14 and S15 .
For miRNA quantification, a miRNA stem-loop primer and a pair of primers were designed for miRNA reverse transcription and qPCR, respectively. The U6 gene was used as the internal normalization control. MiRNA stem-loop was reverse transcribed with the PrimeScript RT reagent Kit and quantified by qPCR using the SYBR premix Ex Taq qPCR Kit, according to manufacturer’s protocols. Relative gene expression was determined by the 2−ΔΔCt method50 (link)51 (link). The primers used for qPCR are listed in