title: [Improved RT-PCR for detection of PML/RARalpha fusion gene in rapid diagnosis of acute promyelocytic leukemia].
abstract: Detection of the PML/RARalpha fusion gene by RT-PCR in acute promyelocytic leukemia (APL) blasts is not only critical to commence promptly the specific therapy with all-trans retinoic acid (ATRA) or arsenic trioxide (As(2)O(3)), but also essential for the definition of PML breakpoint type and subsequent monitoring of minimal residual disease (MRD). The current PML/RARalpha amplification techniques with conventional nested PCR are laborious and time consuming, which fails to meet the requirements for rapid diagnosis of APL in clinical practice. Therefore, an easily handled RT-PCR methodology for the rapid and accurate amplification of PML/RARalpha fusion transcripts is needed. A modified one round RT-PCR protocol was described with a few variations which includes rapid extraction of high quality cellular total RNA, cDNA synthesis with random hexamer and M-MLV reverse transcriptase, optimal concentrations of MgCl(2) (1 mmol/L), PCR primers (0.4 micro mol/L) and Taq polymerase (0.01 U/ micro l), hot-start procedure, and concomitant amplification of PML/RARalpha fusion gene and RARalpha internal control under the identical thermocycle parameters. The results in 40 patients with newly diagnosed APL showed that the improved RT-PCR protocol allowed the rapid detection of PML/RARalpha fusion gene and the accurate discrimination of its transcript types, and simultaneous amplification of RARalpha internal control under the identical program in less than 6 hours. There were no false positive or negative results found with the assay. In conclusion, the assay reported here is proved to be a simple, easily handled, and highly specific procedure for the diagnosis of APL cases, particularly those requiring such urgent therapeutic intervention as ATRA or As(2)O(3) and meriting its further application in APL management.
authors.affiliation: Department of Hematology, Second Hospital, Hebei Medical University, Shijiazhuang 050000, China.
authors.forename: Ze-Lin
authors.initials: ZL
authors.lastname: Liu
authors.forename: Jian-Min
authors.initials: JM
authors.lastname: Luo
authors.forename: Fu-Xu
authors.initials: FX
authors.lastname: Wang
authors.forename: Xue-Jun
authors.initials: XJ
authors.lastname: Zhang
authors.forename: Xing-Yan
authors.initials: XY
authors.lastname: Du
authors.forename: Li
authors.initials: L
authors.lastname: Yao
authors.forename: Zuo-Ren
authors.initials: ZR
authors.lastname: Dong
mesh_terms: D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000175:diagnosis* / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein