QUANTIFICATION OF CYTOKINE MESSENGER-RNA EXPRESSION BY RT-PCR AND ELECTROCHEMILUMINESCENCE

Variable gene expression constitutes a major mechanism for controlling cell development and cell function. To investigate these changed mRNA levels, a sensitive and quantitative assay is required. We describe a quick and easy method to quantify specific mRNAs by a combination of PCR and an electrochemiluminescent (ECL) detection of the amplified pr oducts. Total cellular RNA is reverse-transcribed and amplified with a biotinylated forward primer and a Tris (2,2'-bipyvidine) ruthenium (I I) (TBR)-labeled reverse primer. The amplification product is captured on streptavidin-coated paramagnetic beads and quantified by ECL detec tion using the QPCR system 5000. The results can be converted to quant itative values with an external standard curve. This method permits ac curate and reliable quantitation of cytokine mRNA expression.