Manifold-assisted reverse transcription-PCR with real-time detection for measurement of the BCR-ABL fusion transcript in chronic myeloid leukemia patients

Background: BCR-ABL fusion mRNA expression in bone marrow or peripheral blo od can be used as a measure of minimal residual disease in patients with ch ronic myeloid leukemia (CML). Methods: We used an oligo(dT)-coated manifold support to capture the mRNA d irectly from the cell lysate. After reverse transcription, the cDNA was elu ted from the manifold support, and BCR-ABL and GAPDH mRNAs were quantified in real time using the TaqMan fluorogenic detection system. Results: The detection limit of the method was one positive K562 cell among 10(5) negative cells. GAPDH was chosen as a reference gene based on the lo w variation between samples from different stages of the disease and the lo w signal in the absence of reverse transcription. The day-to-day variation of the method (CV) was 32%. In 43 blood samples from 13 CML patients, mRNA quantification agreed well with cytogenetic data. Conclusions: The proposed procedure constitutes a reproducible and sensitiv e BCR-ABL mRNA quantification method and is suitable to monitor minimal res idual disease in CML patients. (C) 2000 American Association for Clinical C hemistry.