J Clin Pathol Qual Control.
1997 Dec;19(2):273-279.
Detection of Major bcr-abl mRNA Using One Step RT-PCR
- Affiliations
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- 1Department of Clinical Pathology, Catholic University Medical College, St. Mary's Hospital, Seoul, Korea.
- 2Clinical Research Institute, Catholic University Medical College, St. Mary's Hospital, Seoul, Korea.
Abstract
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BACKGROUND: Chronic myelogenous leukemia (CML) is characterized by fusion of bcr and abl genes from which major bcr-abl mRNA is generated. To detect major bcr-abl mRNA, reverse transcription-polymerase chain reaction (RT-PCR) with two-step synthesized cDNA to another tube and do PCR. In this technique, we have to transfer synthesized cDNA to another tube and do PCR. In this study, we tried to detect major bcr-abl mRNA using one step RT-PCR by omission of transferring cDNA to another tube before PCR, and to compare the results to that of classical two step RT-PCR.
METHODS
For the one-step RT-PCR, the isolated RNAs from 52 CML patients (36 cases without treatment and 16 cases after bone marrow transplantation (BMT)) were amplified using Titan(TM) One Tube RT-PCR system (Boehringer Mannheim, Germany). To compare the results of the two techniques, the bone marrow aspirates of 20 cases (10 CML chronic phase, 5 CML post-BMT, 5 healthy normal adults) and the serially diluted RNAs up to 1:10,000 were amplified by both techniques at the same time.
RESULTS
In comparision studies, one post-BMT case showed major bcr-abl mRNA only by one step RT-PCR. The other 14 CML cases showed major bcr-abl mRNA and the 5 cases of negative controls did not show major bcr-abl mRNA by both methods. Both techniques detected major bcr-abl mRNA in serially diluted RNAs upto the 1:10,000, but more distinctive band are observed in the one-step RT-PCR than in the two-step RT-PCR, Using one-step RT-PCR, major bcr-abl mRNA was detected in all 36 CML cases without treatment, and mRNA was also found In six of 16 post-BMT cases. Among the 36 CML cases without treatment which revealed major bcr-abl mRNA, 11 (30.6%) cases showed the splice type I (b2a2) and 25 (69.4%) cases showed the splice type II (b3a2).
CONCLUSIONS
One-step RT-PCR technique was highly sensitive and convenient method for the detection of major bcr-abl mRNA.
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