DEVELOPMENT OF COMPETITIVE PCR AND THE QPCR SYSTEM-5000 AS A TRANSCRIPTION-BASED SCREEN

We describe the use of the quantitative PCR (QPCR) system 5000 (Perkin -Elmer) and competitive PCR in a simple and reproducible assay format for use in establishing a screen for the discovery of compounds that a ffect gene regulation. insulin-like growth factor 1 (IGF-1) mRNA was c hosen as an initial target to test the sensitivity and reproducibility of the QPCR System 5000 in the quantitation of PCR products generated in competitive PCR reactions. We found that with the use of sequence- specific probes, the QPCR 5000 could be used easily to distinguish bet ween internal standard (IS) and wild-type products in PCR reactions. W e were able to detect as little as twofold changes in cDNA amounts by using dilutions of total rat liver cDNA as a source of IGF-1 message. The QPCR system 5000 could be used to analyze 24 competitive PCR react ions (48 samples), single determinations, in similar to 1 hr. The flex ibility, automation, and sensitivity of the QPCR System 5000 makes It a useful tool to measure the transcriptional regulation of various mRN As.