Quantification of cytokine messenger RNA in transfected human T cells by RT-PCR and an automated electrochemiluminescence-based post-PCR detection system

Abstract

A fast method is reported for the precise and accurate quantification of cytokine mRNA, based on a quantitative polymerase chain reaction (PCR) assay. Post-PCR detection of the amplification products is achieved using an automated electrochemiluminescent (ECL) detection system. The target is amplified using a biotinylated forward and a tris(2,2'-bipyridine)ruthenium (II) (TBR)-labeled reverse primer. The amplification products are then captured on streptavidin coated paramagnetic beads and quantified by measuring the ECL signal of the TBR label. The results 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 standard. 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 genes.