title: Establishment of real-time TaqMan-fluorescence quantitative RT-PCR assay for detection and quantitation of ten kinds of porcine inflammation markers mRNA.
abstract: Real-time PCR assays based on TaqMan probes for detection of porcine inflammation markers including interferon-α (IFN-α), interferon-β (IFN-β), 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 × 10(1)copies/μl, 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 β-actin in swine testicle (ST) cells transfected with polyinosinic: polycytidylic acid (poly (I:C)). The results showed that the transcription levels (mRNA) of IFN-α, IFN-β, RIG-1 and IRF-7 were up-regulated in ST cells transfected with poly (I:C), 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.
authors.affiliation: Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, China.
authors.forename: Wencheng
authors.initials: W
authors.lastname: Lin
authors.forename: Zhongtian
authors.initials: Z
authors.lastname: Wang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Qiu
authors.forename: Qinfang
authors.initials: Q
authors.lastname: Liu
authors.forename: Shangjin
authors.initials: S
authors.lastname: Cui
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D002478:Cells, Cultured; D005453:Fluorescence; D007249:Inflammation / Q000175:diagnosis* / Q000473:pathology; D008297:Male; D057089:Pathology, Molecular / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000473:pathology; D013737:Testis / Q000473:pathology; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger
keywords: Cytokine; Inflammation markers; Porcine; Real-time PCR; Type I interferon