Evaluation of housekeeping genes for normalizing real-time quantitative PCR assays in pig skeletal muscle at multiple developmental stages

• We examine 16 candidate reference genes in pig skeletal muscle development.
• DRAP1 and RNF7 are suitable as reference genes in 11 developmental stages in Yorkshire pig.
• DRAP1 and WSB2 are suitable as reference genes in 11 developmental stages of Tongcheng pig.

Quantitative PCR (qPCR) requires a constantly expressed housekeeping gene as an internal control, the expression of which is similar in different biological samples. In the present study, we evaluated the applicability and compared the consistency of the gene expression of 16 reference genes, i.e., 10 common and 6 candidate genes, through qPCR assays in pig skeletal muscles at multiple developmental stages. The stability of these 16 potential reference genes was investigated using the geNorm and NormFinder methods. Our results indicated that DRAP1 and RNF7 were the most appropriate combination to normalize gene expression in the Yorkshire samples, the combination of DRAP1 and WSB2 were appropriate in the Tongcheng samples, H3F3A and DRAP1 in prenatal periods, DRAP1 and RNF7 in postnatal periods, and the combination of DRAP1 and WSB2 was most suitable for accurate normalization in whole samples. This result provides a strong basis for similar studies in pigs that explore the molecular mechanisms underlying skeletal muscle development.