tetano
Editor, Senior Moderator
J Virol Methods. 2014 May 2. pii: S0166-0934(14)00174-8. doi: 10.1016/j.jviromet.2014.04.014. [Epub ahead of print]
TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses.
Carolan LA1, Butler J2, Rockman S3, Guarnaccia T4, Hurt AC1, Reading P1, Kelso A1, Barr I1, Laurie KL5.
Author information
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.
Copyright ? 2014. Published by Elsevier B.V.
KEYWORDS:
Cytokine, Ferret, Gene, Real time RT-PCR, Taqman
PMID:
24797460
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/24797460
TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses.
Carolan LA1, Butler J2, Rockman S3, Guarnaccia T4, Hurt AC1, Reading P1, Kelso A1, Barr I1, Laurie KL5.
Author information
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.
Copyright ? 2014. Published by Elsevier B.V.
KEYWORDS:
Cytokine, Ferret, Gene, Real time RT-PCR, Taqman
PMID:
24797460
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/24797460