TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

Louise A Carolan; Jeff Butler; Steve Rockman; Teagan Guarnaccia; Aeron C Hurt; Patrick Reading; Anne Kelso; Ian Barr; Karen L Laurie

doi:10.1016/j.jviromet.2014.04.014

TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

J Virol Methods. 2014 Sep 1:205:38-52. doi: 10.1016/j.jviromet.2014.04.014. Epub 2014 May 4.

Authors

Louise A Carolan¹, Jeff Butler², Steve Rockman³, Teagan Guarnaccia⁴, Aeron C Hurt¹, Patrick Reading¹, Anne Kelso¹, Ian Barr¹, Karen L Laurie⁵

Affiliations

¹ 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.
² 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.
³ 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.
⁴ 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.
⁵ 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.

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.

Keywords: Cytokine; Ferret; Gene; Real time RT-PCR; TaqMan.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adaptive Immunity*
Animals
Cytokines / genetics*
Disease Models, Animal
Female
Ferrets
Genes, Essential / genetics
Humans
Immunity, Innate*
Leukocytes / immunology
Male
Mitogens / immunology
Orthomyxoviridae / immunology*
Real-Time Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction / methods*

Substances

Cytokines
Mitogens