Screening and validation of reference genes for qRT-PCR of bovine skeletal muscle-derived satellite cells

Abstract

AbstractThe accuracy of sixteen commonly used internal reference genes was assessed in skeletal muscle-derived satellite cells of Qinchuan cattle at different stages of proliferation and induction of differentiation to determine the most suitable ones. Quantitative real-time PCR and three commonly used algorithmic programs, GeNorm, NormFinder and BestKeeper, were used to evaluate the stability of expression of the candidate internal reference genes (GAPDH, ACTB, PPIA, LRP10, HPRT1, YWHAZ, B2M, TBP, EIF3K , RPS9, UXT, 18S rRNA, RPLP0, MARVELD, EMD and RPS15A) in skeletal muscle-derived satellite cells at 0, 12, 24, 36 and 48 h of growth and after differentiation for 0, 2, 4, 6 and 8 days. The expression of two satellite cell marker genes, CCNA2 and MYF5, was used for validation analysis. The results of the software analyses showed that GAPDH and RPS15A were the most stable reference gene combinations during in vitro proliferation of bovine skeletal muscle-derived satellite cells, RPS15A and RPS9 were the most stable reference gene combinations during in vitro induction of differentiation of the cells, and PPIA was the least stable reference gene during proliferation and differentiation and was not recommended. This study lays the foundation for the selection of reference genes for qRT-PCR during the proliferation and induction of differentiation of bovine skeletal muscle-derived satellite cells.