Quantitative RT-PCR combined with time-resolved fluorometry for determination of BCR-ABL mRNA

Abstract

Abstract A microtiter well-based quantitative reverse transcriptase-PCR assay for determination of BCR-ABL mRNA, which relies on coamplification of the target with an RNA internal standard (IS), was developed. The hapten digoxigenin (Dig) is incorporated during PCR. Target RNA and IS contain identical primer recognition sites and generate same-sized amplification products distinguishable by hybridization with probes specific to the molecules' central part. The hybrids are determined with an anti-Dig-alkaline phosphatase conjugate with fluorosalicylphosphate as substrate. Fluorescent complexes of fluorosalicylate-Tb(III)-EDTA are measured by time-resolved fluorometry. The ratio of fluorescence values for target and IS is linearly related to initial target RNA in the range of 1000 to 200000 molecules. Samples containing K562 total RNA amidst 1 microgram of RNA from normal cells give fluorescence ratios that are linearly related to 30-10000 K562 cells. CVs for 30, 200, and 900 K562 cells are approximately 11%.