Detection Of PML-RARa Transcripts In Acute Promyelocytic Leukemia By Real-time Quantitative Reverse Transcriptase Polymerase Chain Reaction

Objective: To establish a real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method for detection of PML-RARa fusion gene in acute promyelocytic leukemia (APL) and to explore the relationship between the expression level of the PML/RARa fusion trscript and the clinical status and efficacy of the therapy in APL.Methods: The conventional RT-PCR was used to amplify PML/RARa gene from cultured NB4 cells.Standard curves were constructed by performing modified real-time PCR on standard template after 10-fold serial dilutions of cells cDNA .The sensitivity ,stability and repetitiveness of this method was determined. Eight samples of bone marrow from 4 APL patients were collected before and after treatment .Another patient's PML/RARa mRNA levels at diagnosis ,relapse different time intervals were observed.Results: The sensitivity of real-time quantitative RT-PCR for detecting PML/RARa fusion gene was about 10^-5 μ g cDNA from NB4 cells. Repeatability and reproducibility of the method were satisfying as intra- and inter-assay coefficient variation (CV) were 2.1%, 3.8%. The PML/RARa fusion gene expression levels of 4 patients at diagnosis were 1884 ,5533 ,1803, 4677 and the median gene expression level was 3475.After ATRA plus chemotherapy , The PML/RARa fusion gene expression levels were reduced to 40, 135, 79, 229 and the median gene expression level was 121. Another patient's PML/RARa gene copy was 8600 at diagnosis . After 4 months of therapy the gene copy number was 730 ,although he...