Competitive RT-PCR ELISA: a rapid, sensitive and non-radioactive method to quantitate cytokine mRNA

We have developed a non-radioactive method to quantitate precisely levels of gene expression. This method is based on RT-PCR (reverse transcriptase-polymerase chain reaction) with an RNA competitor, followed by the covalent capture of the amplified DNA onto the wells of microtiter plates, and the quantitation of the PCR product by oligonucleotide hybridization and ELISA (enzyme-linked immunosorbent assay). The assay can reproducibly detect 1 zeptomole mRNA. The assay was successfully used to quantitate mRNA levels of the T cell derived cytokines interleukin-2, interleukin-4 and interferon-γ in resting and stimulated human lymphocytes. Because it is performed in a microtiter ELISA format, this rapid, sensitive and non-radioactive method should facilitate measurements of gene expression, particularly in large clinical studies.