Selection of reference genes for gene expression studies in porcine hepatic tissue using quantitative real-time polymerase chain reaction

Quantitative real-time polymerase chain reaction (RT-qPCR) has become an indispensable technique for accurate determination of gene expression in variety of samples. Accurate and reliable quantification, however, depends on a proper normalization strategy. Normalization with multiple uniformly expressed reference genes is becoming the standard, although the most suitable reference genes dependent on the used experimental factors as well as the tissue or cell type studied. In this study, the stability of various reference genes was investigated in porcine hepatic tissue. The study was conducted on Polish Large White, Polish Landrace, Pietrain, Pulawska and Duroc pigs slaghtered at different ages. Nine reference genes (ACTB, B2M, GAPDH, HPRT1, RPL13A, SDHA, TBP, TOP2B and YWHAZ) were investigated on 180 mRNA samples of porcine hepatic tissue. Based on geNorm and NormFinder analysis, three most stable (HPRT1, TOP2B and TBP) and three moderately (GAPDH, ACTB and SDHA) stable reference genes were identified. The study provides a new panel of reference genes for normalization of the expression of a gene of interest in porcine liver tissue. It is concluded that the use of a single gene for normalization may lead to relatively large errors, so it is important to use multiple control genes based on a survey of potential reference genes applied to gene expression profiling studies of candidate genes for economic traits in pigs.