| Popis: |
Introduction: The multidrug resistance protein 1 (MDR1) has the capacity to extrude chemotherapeutics and has been implicated in treatment failure in acute myeloid leukemia (AML). Previous methods for determination of MDR1 expression have included dye exclusion, demonstration of P-glycoprotein by flow cytometry and/or immunohistochemistry, and molecular polymerase chain reaction (PCR)-based assays for RNA expression. However, these assays have either proven difficult to standardize or tedious to perform. We have therefore designed a real-time quantitative (RQ)-PCR based assay measuring MDR1 gene expression and validated it in AML patients by direct comparison with a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay. Patients and methods: Bone marrow or peripheral blood from 101 AML patients diagnosed (1987–96) at our department were assessed for quantitative expression of MDR1 employing TaqMan RQ-PCR. These data were compared with results obtained by a semi-quantitative competitive PCR assay employing an artificial internal RNA construct. Results: While the RQ-PCR method was able to determine MDR1 gene expression in a continuous fashion over five logs, the semi-quantitative PCR only yielded data in a discontinuous fashion and over four logs at best. Compared with the MDR1 positive and negative cell lines 8226 DOX40 and REH AML cells exhibited variation of 10 PCR cycles, equivalent to a 1000-fold difference. A significant correlation was observed between the two methods, Spearman's correlation coefficient = –0.502, P-value = 10−5. Conclusion: We conclude that, RQ-PCR is a novel methodology, which enables sensitive and quantitative measurement of MDR1 gene expression. This assay is moreover suitable because of its high throughput for longitudinal follow-up and large number of patients. |
