Selection of Reference Genes for Gene Expression Studies in Porcine Whole Blood and Peripheral Blood Mononuclear Cells under Polyinosinic:Polycytidylic Acid Stimulation

Wang, Jiying; Wang, Yanping; Wang, Huaizhong; Hao, Xiaojing; Wu, Ying; Guo, Jianfeng

There are a number of programs that allow assessment of multiple reference genes, such as geNorm (Vandesompele et al., 2002) , NormFinder (Andersen et al., 2004) and Bestkeeper (Pfaffl et al., 2004) . But to date, there is no consensus on which method should be used to examine reference gene expression stability. In particular, Radoni et al. (2005) suggested that ranking lists of the genes tested was tool-dependent. A comparison of different algorithms of reference gene selection allows a better evaluation of the most reliable controls and reduces the risk of artificial selection of co-regulated transcripts. Therefore, in the present study, we used two statistical approaches, geNorm and NormFinder, to evaluate the gene expression stability of the six reference genes, and also compare the results obtained using these two programs. Based on the geNorm programs, the most stable reference genes in both whole blood and PBMC were PPIA and B2M. Combination of the two genes can obtain rather small M values for the samples of whole blood (0.299) and PBMC (0.490). As experimental data from the geNorm developers have shown that stably expressed genes typically exhibit mean M values lower than 0.5 in relatively homogeneous sample panels (Hellemans et al., 2007) . Thus, the combining of the two genes, PPIA and B2M, is appropriate for normalization of gene expression of whole blood and PBMC. On the other hand, based on the NormFinder, TBP was ranked as the most stable reference gene in both whole blood and PBMC, while genes B2M and PPIA were the second and third ones. This discrepancy is probably due to the different algorithms used in the two programs. Though the most stable genes were different, the first three most stable genes were the same as evaluated by the two programs.