Rd. Hockett et al., SIMULTANEOUS QUANTITATION OF MULTIPLE CYTOKINE MESSENGER-RNAS BY RT-PCR UTILIZING PLATE BASED EIA METHODOLOGY, Journal of immunological methods, 187(2), 1995, pp. 273-285
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.