QUANTITATIVE RT-PCR COMBINED WITH TIME-RESOLVED FLUOROMETRY FOR DETERMINATION OF BCR-ABL MESSENGER-RNA

A microtiter well-based quantitative reverse transcriptase-PCR assay f or determination of BCR-ABL mRNA, which relies on coamplification of t he target with an RNA internal standard (IS), was developed. The hapte n digoxigenin (Dig) is incorporated during PCR Target RNA and IS conta in identical primer recognition sites and generate same-sized amplific ation products distinguishable by hybridization with probes specific t o the molecules' central part. The hybrids are determined with an anti -Dig-alkaline phosphatase conjugate with fluorosalicylphosphate as sub strate. Fluorescent complexes of fluorosalicyIate-Tb(III)-EDTA are mea sured by time-resolved fluorometry. The ratio of fluorescence values f or target and IS is linearly related to initial target RNA in the rang e of 1000 to 200 000 molecules. Samples containing K562 total RNA amid st 1 mu g of RNA from normal cells give fluorescence ratios that are l inearly related to 30-10 000 K562 cells. CVs for 30, 200, and 900 K562 cells are similar to 11%.