Quantitative comparison of NASBA-ELISA and RT-PCR-ELISA sensitivities for measurement of the BCR-ABL genes fusion transcript in CML patients

ABSTRACT

Quantitative comparison of NASBA-ELISA and RT-PCR-ELISA sensitivities for measurement of the BCR-ABL genes fusion transcript in CML patients Nazemi A1, Sadeghizadeh M2, Forouzandeh Moghaddam M3, Javadi Gh4, Hashemi M5 1 Student of PhD of Molecular Genetics, Department of Biology, Islamic Azad University, Science and Research Campus, Tehran, Iran. 2Associate Professor, Department of Genetics, Tarbiat Modarres University, Tehran, Iran. 3 Associate Professor, Department of Medical Biotechnology, Tarbiat Modarres University, Tehran, Iran. 4 Associate Professor, Department of Biology, Islamic Azad University, Science and Research Campus, Tehran, Iran. 5 Assistant Professor,Department of Molecular Genetics, Islamic Azad University, Tehran Medical Branch, Tehran, Iran. 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 [q34; q11]. 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. 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. 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. While NASBA technique does not need thermal cycler PCR but has less sensitivity than RT-PCR and is not suitable for quantitative assessment