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 affect gene regulation. Insulin-like growth factor 1 (IGF-1) mRNA was chosen 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 between internal standard (IS) and wild-type products in PCR reactions. We 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 reactions (48 samples), single determinations, in approximately 1 hr. The flexibility, automation, and sensitivity of the QPCR System 5000 makes it a useful tool to measure the transcriptional regulation of various mRNAs.
Wilkinson
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Allelix Biopharmaceuticals, Inc., Mississauga, Ontario, Canada.
Cheifetz
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De Grandis
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D000818:Animals; D001331:Automation; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D005786:Gene Expression Regulation; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I