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Biotechnical Methods Section (BTS)

Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients

Abstract

Real-time RT-PCR has great advantages for estimating transcript levels in a variety of situations. These include relative rapid assay times (hours), reliability and ease of performing replicate analyses. In contrast, competitive PCR is a very labor-intensive procedure requiring a few days to generate useful data. We compared the same samples from CML patients by both methods. Importantly, we used the Bcr-Abl junction plasmid DNA, which is used as a competitor in the manual competitive PCR assay, to generate a standard curve for the real-time assay. This permitted reporting the real-time data as the number of BCR-ABL transcripts per μg of total RNA, which is the same format used for the competitive PCR assay. In this study, a total of 435 peripheral blood and marrow samples from 285 CML patients were analyzed by RT-PCR; these patients were undergoing therapy by STI-571, interferon, and bone marrow transplantation treatment. Most samples also had assay values for the Philadelphia chromosome (Ph), FISH and Western blotting for the Bcr-Abl oncoprotein. Our findings indicated that the real-time assay was less sensitive than the manual competitive RT-PCR assay (t = 5.118; P < 0.001). Of interest, the transcript levels in cell line mixtures with various ratios of K562/KG-1 (BCR-ABL positive/negative) cells were also significantly higher with the competitive RT-PCR assays than real-time RT-PCR, except for levels of BCR-ABL below 200 transcripts per μg of RNA. In both patient and cell line experiments, dividing the BCR-ABL transcripts by the total ABL transcripts virtually eliminated the difference between real-time BCR-ABL transcript values and quantitative competitive BCR-ABL transcript values, indicating that both BCR-ABL and ABL transcripts were underestimated by the real-time assay. In addition, the increased sensitivity of the nested, competitive RT-PCR was readily apparent in patients with minimal residual disease, which by the real-time were negative in the majority of patients but were positive by nested, competitive RT-PCR in 44.6% (n = 29) of samples analyzed (n = 65). These findings indicate that real-time RT-PCR, when normalized for the total ABL transcripts, can be used to monitor CML patients during therapy, but we suggest that nested, competitive RT-PCR be used to determine BCR-ABL/ABL transcript ratios at low transcript values or especially when real-time analyses are negative.

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Acknowledgements

We would like to thank Dr Goldman for the competitor plasmids, and Dr Feng Lin for advice and suggestions. In addition, we would like to thank Dr Ke Si for help in statistical analysis and we acknowledge expert technical assistance of Qing Wang and Jialing Xu. This research was supported by grants from NIH (CA49639) and the Hendricks Foundation.

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Guo, J., Lin, H., Kantarjian, H. et al. Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients. Leukemia 16, 2447–2453 (2002). https://doi.org/10.1038/sj.leu.2402730

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  • DOI: https://doi.org/10.1038/sj.leu.2402730

Keywords

  • real-time RT-PCR
  • quantitative competitive RT-PCR
  • BCR-ABL transcripts
  • chronic myeloid leukemia

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