Competitive reverse-transcriptase polymerase chain reaction without an artificial internal standard

Analytical Biochemistry
M E ZenilmanA R Shuldiner

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...Continue Reading

Citations

Jul 24, 1998·Clinical Chemistry and Laboratory Medicine : CCLM·C OrlandoM Pazzagli
Sep 13, 2003·Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc·J R SmileyD J Jackwood
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