Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms

Abstract

Real-time quantitative PCR (RT-qPCR) is a widely applied technique for relative quantification of gene expression. In this context, the selection of a suitable reference gene (RG) is an essential step for obtaining reliable and biologically relevant RT-qPCR results. The present study aimed to determine the expression stability of commonly used RGs in mouse skeletal muscle tissue. The expression pattern of eight RGs (ACTB, GAPDH, HPRT, YWHAZ, B2M, PPIA, TUBA and 18S) were evaluated by RT-qPCR in different sample groups classified based on genetic background, muscle tissue type, and growth stage, as well as in a C2C12 myoblast cell line model. Five computational programs were included in the study (comparative ΔCq value, NormFinder, BestKeeper, geNorm, RefFinder) to evaluate the expression stability of RGs. Furthermore, the normalization effects of RGs in soleus (SOL) and gastrocnemius (GAS) muscle tissue were evaluated. Collectively, ACTB, HPRT and YWHAZ were shown to be the most stable RGs, while GADPH and 18S were the least stable. Therefore, the combined use of ACTB, HPRT and YWHAZ is recommended for the normalization of gene expression results in experiments with murine skeletal muscle. The results discussed herein provide a foundation for gene expression analysis by RT-qPCR in mammalian skeletal muscle.