Establishment of real-time TaqMan-fluorescence quantitative RT-PCR assay for detection and quantitation of ten kinds of porcine inflammation markers mRNA

Real-time PCR assays based on TaqMan probes for detection of porcine inflammation markers including interferon-alpha (IFN-alpha), interferon-beta (IFN-beta), retinoic acid-inducible gene 1 (RIG-1), toll-like receptor 9 (TLR-9), interferon regulatory factors (IRF-3, IRF-7), Janus kinase (JAK-1, JAK-2), signal transducers and activators of transcription (STAT-1, STAT-2) were established in this study. The results indicated that the established assays were highly specific and sensitive with a detection limit of 1.0 x 10(1) copies/mu l, and coefficient of variations was less than three percent for both inter- and intra-assay. The established assays were used to detect mRNA levels of these inflammation markers and beta-actin in swine testicle (ST) cells transfected with polyinosinic: polycytidylic acid (poly (I:C)). The results showed that the transcription levels (mRNA) of IFN-alpha, IFN-beta, RIG-1 and IRF-7 were up-regulated in ST cells transfected with poly (LC), and the transcription levels (mRNA) of TLR-9, IRF-3, JAK-1, JAK-2, STAT-1 and STAT-2 showed minimal change. The real-time PCR assays established in this study with high specificity, sensitivity and reproducibility could be used to quantify mRNA levels of porcine inflammation markers. (c) 2013 Elsevier B.V. All rights reserved.