Semiquantitative determination of human cytokine mRna expression using TaqMan RT-PCR.
To establish an easy, fast and reliable RT-PCR for the analysis of human cytokine expression, we made use of the recently developed technique of TaqMan PCR. This technique is based on the cleavage of fluorochrome-labelled internal oligodeoxynucleotide probes by the 5'-->3' nuclease activity of Taq DNA polymerase. Measurement of fluorescence intensity during each cycle of the PCR reaction with a Sequence Detection System allows the determination of a threshold cycle at which an increase in fluorescence intensity is first detectable. From these values, a starting amount of template DNA can be calculated. Here, we established specific primers and corresponding internal, fluorogenic probes for the human cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma), and for the constant region of the T-cell receptor beta chain (TCRbeta) and the housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) for normalization of mRNA expression levels. Titrations of the cDNA input showed a strict inverse correlation between the threshold cycles obtained and the starting amount of template. This in turn allowed the generation of a standard curve, and thus quantification of mRNA abundance in cDNA samples. Evaluation of the method using cDNAs from peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or phytohae-magglutinin (PHA) showed basal expression of TNF-alpha and IL-1beta in untreated PBMC while IFN-y was not detectable or only weakly expressed. After stimulation with LPS, a strong induction of IL-1beta and TNF-alpha was measured, while IFN-gamma was induced to a lesser extent. PHA treatment, in contrast, led to an induction of all three cytokines with IFN-gamma being the most prominent. The method has a large dynamic range, requires no post-PCR processing and gives reliable results.
Lang
R
R
Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany.
Heeg
K
K