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Quantitative comparison of NASBA-ELISA and RT-PCR-ELISA sensitivities for measurement of the BCR-ABL genes fusion transcript in CML patients
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Ali Nazemi   1, Majid Sadeghizadeh , Mehdi Forouzandeh Moghaddam , Gholamreza Javadi , Mehrdad Hashemi |
| 1- , Alinazemy@yahoo.com |
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Abstract: (30001 Views) |
Background: Chronic myeloid leukemia (CML) is characterized by neoplastic overproduction of myelocytes and neutrophils. Affected patients have a Philadelphia chromosome which arises following a translocation between long arms of chromosome 9 and 22 (q34q11). This results in abelson murine/breakpoint cluster region (BCR/ABL) fusion. Detection of cells carrying BCR/ABL fusion is extremely important in monitoring response to treatment, remission and relapse in CML patients. In this study, we compared RT-PCR and NASBA techniques to determine quantitatively the number of bcr/abl transcripts.
Materials and Methods: Fusion transcripts were synthesized and RNA was extracted from K562 leukemic cell line. A serial dilution of both fusion transcript and RNA was prepared then sensitivities of both techniques were determined. RT-PCR and NASBA reaction products were labeled using equal ratios of DIG-11-dUTP and DIG-11-UTP respectively. Following denaturation, hybridization reactions were carried out with specific probes. The products were incubated in streptavidin coated microplates. Then, the plates were washed, anti-DIG conjugated with peroxidase added and using ATBS as substrate, enzymatic activity was determined by absorption at 405 nm.
Results: The results showed that specificity of two techniques was equal but RT-PCR-ELISA sensitivity was about 100-fold more than NASBA-ELISA as it could detect 100 pg RNA less than NASBA-ELISA (0.006 versus 0.06 pg RNA). Furthermore, leukemia cell detection precision by RT-PCR-ELISA and NASBA-ELISA was 4 and 400 cells, respectively.
Conclusion: While NASBA technique does not need thermal cycler PCR but has less sensitivity than RT-PCR and is not suitable for quantitative assessment. |
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| Keywords: RT-PCR-ELISA, NASBA-ELISA, BCR/ABL transcript |
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Full-Text [PDF 570 kb]
(3262 Downloads)
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Subject:
Epidemiology
Received: 2006/09/6 | Published: 2008/07/15
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