Quantification of bcr/abl mRNA expression level in leukemia cells by FQ-RT-PCR.
OBJECTIVE
To establish a fluorogenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of bcr/abl mRNA fusion gene expression level in leukemia cells, and provide a useful tool for leukaemia diagnosis and minimal residual disease inspectation.


METHODS
The conventional RT-PCR was used to amplify bcr/abl gene from cultured K562 cells, the quantitative standard template was constructed with A-T clone method. The fluo-rogenic quantitative RT-PCR method by using Applied Biosystems 7700 Sequence Detector for detecting the expression of bcr/abl fusion gene was successfully. The sensitivity, stability and repetitiveness of this method was determined. The peripheral blood samples from 14 CML patients, one of whom before and one month after bone marrow transplantation (BMT) and 4 cases of ALL in the early stages were detected.


RESULTS
The sensitivity of FQ-RT-PCR for detecting bcr/abl fusion gene was about 10(-5) micro g RNA from K562 cell and 10 copies recombined plasmid. The repetition CT value (cycle threshold) and the coefficient variation (CV) among tubes and batches were 2.0% and 3.7%, respectively. The median bcr/abl fusion gene expression level of 14 CML patients was 5.15 x 10(4) copies/ micro g RNA. The products analyzed by electrophoresis showed that 11 cases were b2a2 and 3 cases b3a2. 1.2 x 10(5) copies/ micro g RNA in one CML patient before BMT was changed to 2.3 x 10(3) copies/ micro g RNA one month after BMT. B2a2 was observed in one of the four (25.0%) patients with ALL, and its level of expression was 8.2 x 10(5) copies/ micro g RNA.


CONCLUSIONS
The established FQ-RT-PCR method is sensitive, specific, reliable, accurate and good at repetitiveness. The results expressed in copies were easy for evaluation and comparation. Two different bcr/abl fusion gene form - b2a2, b3a2 can be detected by the method. It can be widely applied to diagnosis and detection of minimal residual disease for CML and some ALL patients.
Gao
Jingsong
J
Daan Gene Diagnosis Center, Sun Yat-sen University, Guangzhou 510089, China.
Tong
Ming
M
He
Yunshao
Y
Nie
Changfu
C
Zhou
Yuanfan
Y
D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D016313:Genes, abl; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl