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 modified oligo(dT) primers (designated RT primers) containing a sample-specific sequence and a common sequence. After equal amounts of the cDNAs were mixed together, a target gene was amplified by competitive PCR with additional primers: 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 different 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 single detection. This system should be useful for sensitive measurement of scarce transcripts from small samples with a limited amount of RNA such as biopsy specimens.
Omori
Y
Y
Department of Virology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo, 108-8639, Japan.
Tanigami
A
A
Sugano
S
S
D000818:Animals; D017931:DNA Primers; D005453:Fluorescence; D015870:Gene Expression; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012333:RNA, Messenger