QUANTIFICATION OF CYTOKINE GENE-EXPRESSION BY COMPETITIVE PCR USING ACOLORIMETRIC ASSAY

Competitive reverse transcription-polymerase chain reaction (RT-PCR) i s a new technique allowing quantification of cytokine gene expression from either experimental or clinical samples, In this assay, a time-co nsuming step is the quantification of amplified products. To improve t his step, we set up a colorimetric assay in which the amplified produc t from either the cDNA or the competitor can be reliably quantified. U sing this approach, which can be completely automatized, up to 320 PCR products can be quantified each day, In this report, we describe the quantification of IL-10 mRNA molecules as compared to that of beta-act in mRNA molecules. The sensitivity of the quantification was 7.7 x 10( 7) molecules for the amplified beta-actin cDNA and the amplified IL-10 cDNA, corresponding to approximately 9.6 pg amplified beta-actin cDNA and 11 pg amplified IL-10 cDNA, respectively. The intra-assay variati on coefficient was <12%. This technique can be readily extended to all cytokines, and it thus allows routine monitoring of cytokine gene exp ression, either from experimental samples or from clinical trials.