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| Quantification of six different PML/RARα isoforms in patients with acute promyelocytic leukemia using quantitative real time PCR |
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HAN Lan-xiu1,2， LIN Jiang1， XU Xin2， QIAN Jun1， ZHOU Jian-bo2， WANG Ya-li1 |
| （1. Department of Hematology， the Affiliated People′s Hospital of Jiangsu University， Zhenjiang Jiangsu 212002； 2. Department of Laboratory，the Affiliated Jiangyin Hospital， College of Medicine， Southeast University， Jiangyin Jiangsu 214400， China） |
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Abstract
Objective: To establish a quantitative real time PCR(RQ-PCR) method for detecting the six different PML/RARα isoforms(bcr1/2,P4R3,P46R3,bcr3,P2R3) in acute promyelocytic leukemia (APL) patients, and evaluate the specificity of isoform-specific RQ-PCR. Methods: Specific primers and probe of different isoforms were designed and isoform-specific RQ-PCR method for detecting each isoform was established. To evaluate the utility of this assay, bone marrow samples from 10 APL patients were detected. Results: In repeated tests, maximal sensitivity of 10 copies/μL for each isoform-specific RQ-PCR was obtained, however, the reproducible maximal sensitivities achieved 100 copies/μL. In the normal controls and the no-template control, any amplified fluorescent signals were not detected. Each isoform-specific RQ-PCR could not amplify other isoforms. In five bcr1-positive cases and one bcr2positive case, the expression level of bcr1/2, P4R3 and P46R3 isoforms was 7.71%-89.71% (median 13.70%), 0.71%-16.26% (median 4.48%),3.57%-14.09% (median 6.33%), respectively. In four bcr3-positive cases, the expression level of bcr3 and P2R3 isoforms was 21.43%-197.04% (median 100.69%) and 0.34%-9.07% (median 2.45%). The expression level of bcr3 and P2R3 isoforms significantly decreased in one case achieving complete remission after induction therapy compared to that in initial diagnosis, and significantly increased after relapse. Conclusion: Isoform-specific RQ-PCR method for six PML/RARα isoforms was a sensitive, reliable quantitative assay and could be used in the detection of the six different PM/RARα isoforms of APL and the measurement of minimal residual disease.
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Received: 22 May 2014
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2014, Vol. 24 
