SIMULTANEOUS QUANTITATION OF MULTIPLE CYTOKINE MESSENGER-RNAS BY RT-PCR UTILIZING PLATE BASED EIA METHODOLOGY

Cytokines are small protein hormones produced during an immune respons e that are responsible for mediation and regulation of many aspects of immunity. Measurement of cytokines by several different methods has l ed to a broader understanding of the immune response. This paper descr ibes a sensitive, reproducible, and quantitative RT-PCR assay for the simultaneous measurement of multiple cytokines. The main features of t he methodology are: RNA competitors which control for all aspects of t he process from RNA extraction, through reverse transcription (RT) and PCR amplification; a general cloning vector, pQPCR1, for building RNA competitors that does not require prior analyte cDNA cloning; and ana lysis by plate based EIA. This RT-PCR-EIA system is shown to be more s ensitive than agarose gel electrophoresis followed by EtBr staining, m easuring PCR product in the sub-nanogram range. It also extends the li near dynamic range of detection to a four log fold range of analyte co ncentration. The assay is reproducible, with coefficients of variation (CVs) in the 10-20% range. Moreover, the cloning vector is designed t o accommodate multiple primer templates, thus allowing simultaneous qu antitation of many different analytes from a single RT reaction. The d escribed system is versatile and adapts to numerous analytes.