Affordable Access

Publisher Website

Quantification of ovine cytokine gene expression by a competitive RT-PCR method

Authors
Journal
Journal of Immunological Methods
0022-1759
Publisher
Elsevier
Publication Date
Volume
253
Identifiers
DOI: 10.1016/s0022-1759(01)00375-1
Keywords
  • Quantitative Rt-Pcr
  • Mrna
  • Cytokine
  • Ovine
  • Salmonella

Abstract

Abstract A quantitative competitive RT-PCR method was developed in order to measure IL-1β, IL-4, IL-12, IFNγ, TNFα and G 3PDH mRNA from samples of ovine tissue such as lymph node or spleen. The main advantage of the method relies on the use, for each target sequence, of an internal competitor construct similar to the relevant target, but 4-bp different in size. This competitive strategy is validated by the equivalence of the amplification process, observed separately between competitor DNA and target DNA species. Furthermore, the copy number of each cytokine cDNA is normalized to a fixed copy number of G 3PDH cDNA. The cDNA level of this constitutive gene was effectively shown to remain constant whatever the tissue studied and independently of the experimental conditions used. The accurate and reproducible data obtained permit the application of this quantitative RT-PCR method to measure the sheep cytokine response to Salmonella infection. Early induction of IFNγ mRNA was observed in the draining lymph node 1 day after infection. At the same time, a strong increase of IL-1β mRNA was observed in local and systemic lymphoid organs, suggesting the initiation of the inflammatory response. Finally, the overall results demonstrate the efficiency of the method and its suitability for further studies of the immune response in the ovine species.

There are no comments yet on this publication. Be the first to share your thoughts.

Statistics

Seen <100 times
0 Comments

More articles like this

Quantification of ovine cytokine gene expression b...

on Journal of Immunological Metho... Jul 01, 2001

[Competitive RT-PCR assay for quantification of GP...

on Hunan yi ke da xue xue bao = H... August 2003

Quantification of XPA gene expression levels in hu...

on Mutation Research/Fundamental... Jan 31, 1997
More articles like this..