title: TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses.
abstract: The ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states.
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Victoria, 3000, Australia.
authors.forename: Louise A
authors.initials: LA
authors.lastname: Carolan
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Victoria, 3000, Australia; CSIRO Australian Animal Health Laboratory, East Geelong, 3219, Australia.
authors.forename: Jeff
authors.initials: J
authors.lastname: Butler
authors.affiliation: bioCSL Limited, Parkville, 3052, Australia; Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Victoria, 3010, Australia.
authors.forename: Steve
authors.initials: S
authors.lastname: Rockman
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Victoria, 3000, Australia; Monash University Gippsland, Churchill, 3842, Australia.
authors.forename: Teagan
authors.initials: T
authors.lastname: Guarnaccia
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Victoria, 3000, Australia.
authors.forename: Aeron C
authors.initials: AC
authors.lastname: Hurt
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Victoria, 3000, Australia.
authors.forename: Patrick
authors.initials: P
authors.lastname: Reading
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Victoria, 3000, Australia.
authors.forename: Anne
authors.initials: A
authors.lastname: Kelso
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Victoria, 3000, Australia.
authors.forename: Ian
authors.initials: I
authors.lastname: Barr
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Victoria, 3000, Australia. Electronic address: Karen.laurie@influenzacentre.org.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Laurie
mesh_terms: D056704:Adaptive Immunity; D000818:Animals; D016207:Cytokines / Q000235:genetics*; D004195:Disease Models, Animal; D005260:Female; D005289:Ferrets; D020043:Genes, Essential / Q000235:genetics; D006801:Humans; D007113:Immunity, Innate; D007962:Leukocytes / Q000276:immunology; D008297:Male; D008934:Mitogens / Q000276:immunology; D009975:Orthomyxoviridae / Q000276:immunology*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D016207:Cytokines; D008934:Mitogens
keywords: Cytokine; Ferret; Gene; Real time RT-PCR; TaqMan