A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats

J Immunol Methods. 1994 Dec 28;177(1-2):23-8. doi: 10.1016/0022-1759(94)90139-2.

Abstract

Competitive polymerase chain reaction (PCR) is a sensitive method for quantification of cytokine mRNA expression. Co-amplification of an internal standard serves as control for comparing the efficiency of PCR in different samples. We have developed a novel control fragment for multiple analyses of rat cytokine gene expression containing primers for IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, TNF-alpha, TGF-beta 1, IFN-gamma and MIP-2. Additional primers were incorporated to analyse the content of T cells (CD3), activated T cells (CD25) and housekeeping genes (beta-actin and HPRT). As an example we demonstrate analysis of IL-2 mRNA expression in small pieces of kidney tissue obtained from rats after kidney allotransplantation. The IL-2 expression decreased tenfold during treatment with an anti-rat CD4 monoclonal antibody as compared to untreated animals.

MeSH terms

  • Animals
  • Base Sequence
  • Binding, Competitive
  • Cytokines / genetics*
  • DNA Primers / chemistry
  • Gene Expression
  • Interleukin-2 / genetics
  • Kidney Transplantation*
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • RNA, Messenger / genetics
  • Rats

Substances

  • Cytokines
  • DNA Primers
  • Interleukin-2
  • RNA, Messenger