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 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.
Siegling
A
A
Institute for Medical Immunology, Medical Faculty (Charité), Humboldt University, Berlin, Germany.
Lehmann
M
M
Platzer
C
C
Emmrich
F
F
Volk
H D
HD
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D007376:Interleukin-2 / Q000235:genetics; D016030:Kidney Transplantation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats
D016207:Cytokines; D017931:DNA Primers; D007376:Interleukin-2; D012333:RNA, Messenger