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Clinical Chemistry 49: 1066-1073, 2003; 10.1373/49.7.1066
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(Clinical Chemistry. 2003;49:1066-1073.)
© 2003 American Association for Clinical Chemistry, Inc.

Screening and Quantification of Multiple Chromosome Translocations in Human Leukemia

Run Zhang Shi1,a, Joseph M. Morrissey1 and Janet D. Rowley2

1 Motorola Corporate Laboratories, Ft. Lauderdale, FL 33322.

2 Department of Medicine, University of Chicago, Chicago, IL 60637.

aAddress correspondence to this author at: Section of Hematology/Oncology, Department of Medicine, University of Chicago, 5841 S. Maryland, MC2115, Chicago, IL 60637. Fax 773-702-3002; e-mail rshi{at}medicine.bsd.uchicago.edu.

Background: Characterization of fusion gene transcripts in leukemia that result from chromosome translocations provides valuable information regarding appropriate treatment and prognosis. However, screening for multiple fusion gene transcripts is difficult with conventional PCR and state-of-the-art real-time PCR and high-density microarrays.

Methods: We developed a multiplex reverse transcription-PCR (RT-PCR) assay for screening and quantification of fusion gene transcripts in human leukemia cells. Chimeric primers were used that contained gene-specific and universal sequences. PCR amplification of fusion and control gene transcripts was achieved with use of an excess of universal primers to allow the ratio of abundance of fusion gene to endogenous or exogenous controls to be maintained throughout PCR. Multiplex RT-PCR products analyzed by an ABI 310 Genetic Analyzer were consistent with those of duplex RT-PCR (single analytical sample plus control). In addition, multiplex RT-PCR results were analyzed by an assay using an oligonucleotide microarray that contained probes for the splice-junction sequences of various fusion transcripts.

Results: The multiplex RT-PCR assay enabled screening of >10 different fusion gene transcripts in a single reaction. RT-PCR followed by analysis with the ABI Prism 310 Genetic Analyzer consistently detected 1 fusion-transcript-carrying leukemia cell in 100–10 000 cells. The assay covered a 1000-fold range. Preliminary results indicate that multiplex RT-PCR products can also be analyzed by hybridization-based microarray assay.

Conclusions: The multiplex RT-PCR analyzed by either ABI Prism 310 Genetic Analyzer or microarray provides a sensitive and specific assay for screening of multiple fusion transcripts in leukemia, with the latter an assay that is adaptable to a high-throughput system for clinical screening.




The following articles in journals at HighWire Press have cited this article:


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Clinical Screening of Gene Rearrangements in Childhood Leukemia by Using a Multiplex Polymerase Chain Reaction-Microarray Approach
Clin. Cancer Res., November 15, 2003; 9(15): 5620 - 5629.
[Abstract] [Full Text] [PDF]


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