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

All qRT-PCR were carried out using LightCycler 480 SYBR Green I Master on Roche LightCycler 480 instrument following the manufacturer's guidelines. Prior to performing the expression stability assay for all the primers of the six candidate genes, we carried out melting curve analyses to detect their specificity, and then calculated the PCR amplification efficiencies of every primer using a standard curve derived from a pooled cDNA mixture serially diluted 4-fold over five measuring points. In the expression stability assay, all the samples were amplified in duplicate. The PCR reaction consisted of 10 L Blue-SYBR-Green mix, 1 L forward and reverse primers (10 pM/L), 7 L distilled water, and 1 L of cDNA in a total volume of 20 L. The thermal cycling conditions were 3 min at 95C, followed by 40 reaction cycles (10 s at 95C and 20 s at 60C). The second derivative maximum algorithm included within the instrument software was used to determine cycle threshold (Ct) values for each reaction.