title: Development and validation of a SYBR Green real-time PCR assay for rapid and quantitative detection of goose interferons and proinflammatory cytokines.
abstract: Real time quantitative polymerase chain reaction (RT-qPCR) based on SYBR-Green I binding is a quick, reliable, and easy method for analyzing small amounts of mRNA. Viral pathogens are recognized at the time of infection by pattern recognition receptors; thus, the inflammatory cytokines (IL1β, IL6, and IL18) and antiviral cytokines (IFNα, IFNγ) are secreted by innate immune cells and induced to respond to the pathogens. The objective of this study was to develop an effective and sensitive RT-qPCR assay for the rapid and accurate quantification of goose cytokines: IFNα, IFNγ, IL1β, IL6, and IL18. Subsequently, the established methods were employed to detect the immune response in agonist-stimulated goose spleen cells in vitro. These data indicated that the established RT-qPCR is a reliable method for determining relative gene expression. The results revealed that Imiquimod led to the significant upregulation of goose IFNα (P < 0.01), IFNγ (P < 0.01), IL1β (P < 0.01), IL6 (P < 0.01), and IL18 (P < 0.05). The established methods are important for scientific research and clinical applications, which require rapid and accurate results in a short period of time. The technique can potentially be used in the further research of goose molecular immunology, which will help us understand the interactions between hosts and pathogens.
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China sophia_cs@163.com.
authors.forename: Shun
authors.initials: S
authors.lastname: Chen
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China.
authors.forename: Yulin
authors.initials: Y
authors.lastname: Qi
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China.
authors.forename: Mingshu
authors.initials: M
authors.lastname: Wang
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China.
authors.forename: Renyong
authors.initials: R
authors.lastname: Jia
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China.
authors.forename: Dekang
authors.initials: D
authors.lastname: Zhu
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China.
authors.forename: Mafeng
authors.initials: M
authors.lastname: Liu
authors.affiliation: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China.
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China.
authors.forename: Xiaoyue
authors.initials: X
authors.lastname: Chen
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China.
authors.forename: Anchun
authors.initials: A
authors.lastname: Cheng
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D030161:Avian Proteins / Q000187:drug effects / Q000235:genetics*; D052160:Benzothiazoles; D003959:Diamines; D005777:Geese / Q000235:genetics*; D015870:Gene Expression; D000077271:Imiquimod; D007369:Interferon Inducers / Q000494:pharmacology; D007372:Interferons / Q000187:drug effects / Q000235:genetics*; D007378:Interleukins / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D009930:Organic Chemicals; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*; D013154:Spleen / Q000187:drug effects
chemical_list: D000634:Aminoquinolines; D030161:Avian Proteins; D052160:Benzothiazoles; D003959:Diamines; D007369:Interferon Inducers; D007378:Interleukins; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D007372:Interferons; D000077271:Imiquimod
keywords: cytokines; goose; real time quantitative PCR