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

Up to now, most of the published qRT-PCR results were normalized to either GAPDH or ACTB without experimentally confirming their invariant expression under the experimental conditions described. In our present analyses, these two genes were the least and the third least stable genes among the six candidates. Consistent with our result, some previous studies also reported ACTB and GAPDH were unsuitable as reference genes in whole blood (Brym et al., 2013) and PBMC (Spalenza et al., 2011; Xiang-Hong et al., 2011) and other specific samples (Uddin et al., 2011; Zhang et al., 2012) . ACTB is one of the major components of cytoplasmic microfilaments in eukaryotic cells and plays an important role in diverse cellular functions, while GAPDH not only acts as a component of the glycolytic pathway but also takes part in other processes as well. Both ACTB and GAPDH have sophisticated functions in organisms, and their expression profile might fluctuate according to the corresponding experimental conditions. Our analysis results confirmed, once more, blindly choosing reference genes is no longer acceptable, and authors must accurately validate candidate reference genes before use in gene expression qRT-PCR studies.