Detection of bcr-abl transcript in chronic myelogenous leukemia patients by reverse-transcription-polymerase chain reaction and capillary electrophoresis

Giovanni Martinelli, Nicoletta Testoni, Vittorio Montefusco, Marilina Amabile, Giuseppe Saglio, Emanuela Ottaviani, Carolina Terragna, Francesca Bonifazzi, Antonio De Vivo, Fabrizio Pane, Gianantonio Rosti, Sante Tura

Research output: Contribution to journalArticlepeer-review

Abstract

Background and Objective. Capillary electmphoresis (CE) has become an attractive alternative to SLAB gel analysis for direct and accurate detection of amplified product, and a few cycles of polymerase chain reactions (PCRs) could be sufficient for both quantitative and qualitative analysis. We try to assess: 1) whether CE could be a practical, non-isotopic method for direct detection of the presence of amplified bcr-abl obtained by a reverse transcription (RT)-PCR (qualitative analysis) and 2) whether it is possible to quantify PCR products using a competitive RT-PCR measuring peak areas of CE electropherograms (quantitative analysis). Design and Methods. The two types of bcr-abl chronic myelogenous leukemia (CML) associated transcript products were generated by RT-PCR (qualitative analysis) from 1 μg of total RNA extracted from bone marrow samples of 34 CML patients at diagnosis (median age 47.5; range 18-65; median Sokal's score 0.9; range 0.53-2.78). The PCR products were analyzed by SLAB-gel electrophoresis (SGE) on 2% agarose gels and by CE (128 runs; median 3.3 times for each sample). Furthermore, we assessed the amount of PCR product (quantitative analysis) by a competitive RT-PCR approach and by CE (bcr-abl transcripts were expressed as transcript per μg of total RNA examined). Results. CE separation of PCR products obtained by qualitative RT-PCR showed baseline resolution for the two peaks corresponding to the two typos of bcr-abl Junctions: the b2-a2 type (343 base pairs, 10 patients) was revealed at 9.33 min [standard deviation (5D) = 0.1] and the b3-a2 type (418 base pair, 24 patients) at 10.03 min (SD = 0.25). By quantitative analysis we found that there is great interpatient variability in bcr-abl expression at diagnosis: the median value of the amount of bcr-abl transcript was 78,000 bcr-abl transcript/μg total RNA ranging from 17,300 to 750,000. The amount of bcr-abl transcript at diagnosis was related to the number of blast cells (mean value/28,859 vs. 331,722 in patients with 0% blast cells and >1% blast cells, respectively; p = 0.004) and Sokal's score (mean value 156,865 vs. 408,800 in patients with Sokal's score 1.2, respectively; p = 0.003). Interpretation and Conclusions. Our results confirm that CE analysis offers greater resolution and enhanced sensitivity for detection and quantification of bcr-abl PCR product in the study of this leukemia. Qualitative analysis by CE of bcr-abl product provides a rapid technique (less than 20 min) for the analysis of subnanogram amounts of DNA fragments. CE run times are short, the capillary can be re-used and full automation may be feasible with data acquisition by a computer-controlled step. Competitive/quantitative analysis of bcr-abl as analyzed by CE allowed fewer reactions and more precise quantification.

Original languageEnglish
Pages (from-to)593-601
Number of pages9
JournalHaematologica
Volume83
Issue number7
Publication statusPublished - 1998

Keywords

  • Capillary electrophoresis
  • Chronic myelogenous leukemia
  • RT-PCR

ASJC Scopus subject areas

  • Hematology

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