A NOVEL MULTISPECIFIC COMPETITOR FRAGMENT FOR QUANTITATIVE PCR ANALYSIS OF CYTOKINE GENE-EXPRESSION IN RATS

Competitive polymerase chain reaction (PCR) is a sensitive method for quantification of cytokine mRNA expression. Co-amplification of an int ernal standard serves as control for comparing the efficiency of PCR i n different samples. We have developed a novel control fragment for mu ltiple 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 ge nes (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 tenfo ld during treatment with an anti-rat CD4 monoclonal antibody as compar ed to untreated animals.