Competitive reverse-transcriptase polymerase chain reaction without an artificial internal standard.
Academic Article
Overview
abstract
Advances in our understanding of molecular and cellular physiology necessitate that mRNA levels for specific growth factors and other rare transcripts be measured quantitatively in small samples. Conventional methods such as Northern blot analysis and solution hybridization/ribonuclease protection are not sufficiently sensitive. We now report the theory, development, and validation of a rapid and highly sensitive assay, the RNA/DNA quantitative polymerase chain reaction (RD-PCR), which uses a competitive PCR approach to measure the number of copies of a specific mRNA per cell. Total nucleic acid (RNA and genomic DNA) is isolated from cells in culture. The mRNA of interest is first reverse-transcribed with an oligomer bearing a complementary sequence specific for the mRNA at its 3'-end, and a sequence complementary to an intron of the desired gene at the 5'-end. Competitive PCR is then performed in the presence of the cDNA product and endogenous genomic DNA, with an upstream primer complementary to the exon sequence of the gene of interest, and a downstream primer complementary to the intron sequence that was tagged to the cDNA. The cell's own genomic DNA is thereby used as the internal standard. To control for the efficiency of reverse transcription, a standard curve is used in each assay. The technique was validated by comparing the quantitation of insulin-like growth factor I (IGF-I) mRNA in two human cell lines by RD-PCR and by RNase protection analysis. Both methods gave similar numbers of copies of IGF-I mRNA per cell. For accurate analysis, RNase protection required at least 10(7) cells; RD-PCR required as little as 10(2) cells.(ABSTRACT TRUNCATED AT 250 WORDS)
