MATHEMATICAL CONSIDERATIONS OF COMPETITIVE POLYMERASE CHAIN-REACTION

Reverse transcriptase polymerase chain reaction (PCR) is used frequent ly to monitor gene expression. It is generally regarded as a qualitati ve technique, although refinements have been made to improve quantific ation. The object of this study was to develop competitive PCRs to all ow reliable quantification of the rat T cell cytokines interferon-gamm a (IFN-gamma), interleukin-2 (IL-2) and interleukin-4 (IL-4). Truncate d constructs of cDNA for these cytokines were prepared using appropria te pairs of standard and specially constructed primers designed to all ow subsequent co-amplification of the purified competitor construct an d the target cDNA. A high resolution capillary electrophoresis (CE) sy stem was used for PCR product detection. The performance of the system was compared with a mathematical model that describes and predicts th e exponential nature of the PCR reaction. Go-amplification of the comp etitor and target were achieved, A high level of resolution and accura cy was achieved using CE to detect and quantify the PCR products, The rates of generation of the respective products conformed closely but n ot exactly to the predictions of the mathematical model. The competiti ve PCRs estimated initial numbers of target cDNA within 1.1-5.0-fold r elative to the amount of starting material as assessed by conventional spectrophotometric absorbance prior to dilution and amplification. A convenient and flexible competitive PCR strategy has been developed wi th accurate resolution of products and reliable quantification. Assay variability was far less than biological variability likely to be enco untered in experiments investigating immunological responses in rats o r other animals.