COLORIMETRIC ELISA MEASUREMENT OF SPECIFIC MESSENGER-RNA ON IMMOBILIZED-OLIGONUCLEOTIDE-COATED MICROTITER PLATES BY REVERSE TRANSCRIPTION WITH BIOTINYLATED MONONUCLEOTIDES

In this study, alpha-globin mRNA was captured on plastic plates to whi ch alpha-globin-specific oligonucleotides had been covalently immobili zed; the captured mRNA was immediately reverse-transcribed into cDNA i n the presence of biotinylated dUTP. Addition of alkaline phosphatase- conjugated streptavidin to react with the biotin was followed by addit ion of substrate (p-nitrophenyl phosphate), and the resulting colorime tric development was measured at 405 nm. Because multiple biotin molec ules are incorporated into cDNA during reverse transcription, and beca use synthesized cDNA is more stable than mRNA, we could successfully q uantify by conventional colorimetry the amount of alpha-globin mRNA on the plate within a linear range of 100 pg to 10 ng. This is more sens itive than conventional Northern blotting. Therefore, the present meth od may be applicable to measurements of specific mRNAs in various test materials.