Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs

Abstract

The selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis. However, commonly used reference genes have variable expression levels in developing skeletal muscle. There are few reports that systematically evaluate the expression stability of reference genes across prenatal and postnatal developing skeletal muscle in mammals. Here, we used quantitative PCR to examine the expression levels of 15 candidate reference genes (ACTB, GAPDH, RNF7, RHOA, RPS18, RPL32, PPIA, H3F3, API5, B2M, AP1S1, DRAP1, TBP, WSB, and VAPB) in porcine skeletal muscle at 26 different developmental stages (15 prenatal and 11 postnatal periods). We evaluated gene expression stability using the computer algorithms geNorm, NormFinder, and BestKeeper. Our results indicated that GAPDH and ACTB had the greatest variability among the candidate genes across prenatal and postnatal stages of skeletal muscle development. RPS18, API5, and VAPB had stable expression levels in prenatal stages, whereas API5, RPS18, RPL32, and H3F3 had stable expression levels in postnatal stages. API5 and H3F3 expression levels had the greatest stability in all tested prenatal and postnatal stages, and were the most appropriate reference genes for gene expression normalization in developing skeletal muscle. Our data provide valuable information for gene expression analysis during different stages of skeletal muscle development in mammals. This information can provide a valuable guide for the analysis of human diseases.

Genome-Wide Identification and Evaluation of New Reference Genes for Gene Expression Analysis Under Temperature and Salinity Stresses in Ciona savignyi

“…For the moderate expression target genes, RhoA also with a moderate expression level and highly stable expression profile was a good alternative normalizer. RhoA was also identified as suitable reference gene in neuroendocrine tumors of the lung (Niu et al, 2016), and in porcine skeletal muscle at 26 different developmental stages (Walter et al, 2016). However, we have not found that the other two novel reference genes TMN and MOB are used as internal controls in gene expression analysis.…”

Murine miR-483-3p promotes proliferation and suppresses differentiation of C2C12 cells by targeting SRF

“…All reactions were run in triplicate. miRNA expression was normalized to U6 RNA while the internal control gene for myogenin and Myh1 expression was β-Tubulin (Niu, Yang et al 2016). Relative expression was calculated by the comparative ΔΔCT method, and values were expressed as 2 −ΔΔCT (Livak and Schmittgen 2001).…”

Using RNA-Seq to Identify Reference Genes of the Transition from Brown to White Adipose Tissue in Goats

“…Quantitative PCR (qPCR) technology can quickly and accurately quantify the gene expression at transcriptional levels. The qPCR method has been reported to be more specific and powerful to determine the gene expression at different developmental stages or under different conditions [ 9 , 10 ]. However, the results of qPCR are affected by mRNA integrity and the efficiency of reverse transcription [ 11 ].…”