QUANTIFICATION OF CYTOKINE MESSENGER-RNA IN TRANSFECTED HUMAN T-CELLSBY RT-PCR AND AN AUTOMATED ELECTROCHEMILUMINESCENCE-BASED POST-PCR DETECTION SYSTEM

A fast method is reported for the precise and accurate quantification of cytokine mRNA, based on a quantitative polymerase chain reaction (P CR) assay. Post-PCR detection of the amplification products is achieve d using an automated electrochemiluminescent (ECL) detection system. T he target is amplified using a biotinylated forward and a tris(2,2'-bi pyridine)ruthenium (II) (TBR)-labeled reverse primer. The amplificatio n products are then captured on streptavidin coated paramagnetic beads and quantified by measuring the ECL signal of the TBR label. The resu lts obtained are reproducible and accurate over a wide range (3 orders of magnitude) of concentrations. Quantitative results can be obtained using a standard curve which is generated with a synthetic external s tandard. This technique was applied to quantify tumor necrosis factor- alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin-2 (IL- 2) mRNA levels in human T cells transfected with the corresponding gen es.