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Clin Chem. 2000 Jul;46(7):913-20.

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

Author information

1
Department of Medical Sciences, Uppsala University, 75185 Uppsala, Sweden. gisela.barbany@medsci.uu.se

Abstract

BACKGROUND:

BCR-ABL fusion mRNA expression in bone marrow or peripheral blood can be used as a measure of minimal residual disease in patients with chronic myeloid leukemia (CML).

METHODS:

We used an oligo(dT)-coated manifold support to capture the mRNA directly from the cell lysate. After reverse transcription, the cDNA was eluted 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 low variation between samples from different stages of the disease and the low 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 sensitive BCR-ABL mRNA quantification method and is suitable to monitor minimal residual disease in CML patients.

PMID:
10894833
[Indexed for MEDLINE]
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