Comparative PCR: A simple and sensitive method for quantifying low-abundance mRNA species

To measure low-abundance messenger RNA species comparatively, we developed a simple and highly sensitive quantification method designated comparative PCR. Messenger RNAs from two samples were converted into cDNAs with modifie d oligo(dT) primers (designated RT primers) containing a sample-specific se quence and a common sequence, After equal amounts of the cDNAs were mixed t ogether, a target gene was amplified by competitive PCR with additional pri mers: a gene-specific primer and a primer consisting of the common sequence of the RT primers. The amplified products were visualized by the final PCR , designated fluorescence PCR, with an additional three primers: two differ ent fluorescence-labeled primers consisting of the sample-specific sequence within the RT primers and a nested gene-specific primer. Expression levels of the target gene in the two samples were measured by calculating ratios of two different fluorescence intensities. We could quantify 0.1-0.3 copies of the target mRNA per cell from only 0.5 ng of poly(A)(+) RNA for a singl e detection. This system should be useful for sensitive measurement of scar ce transcripts from small samples with a limited amount of RNA such as biop sy specimens. (C) 2000 Academic Press.