One-tube multiplex RT-PCR of BCR-ABL transcripts in analysis of patients with chronic myeloid leukaemia and acute lymphoblastic leukaemia

We describe a one-tube multiplex reverse transcription polymerase chain rea ction (RT-PCR) assay for the detection of bcr-abl fusion mRNA in analysis o f patients with chronic myeloid leukaemia and acute lymphoblastic leukaemia . The assay provides a quick and reliable method for the detection and anal ysis of chromosome translocations resulting in formation of the fusion prot eins p210 (b3a2/b2a2) and p190 (e1a2). The method is based on the use of ma gnetic beads and sequence-specific reverse transcription primers. By combin ing direct mRNA isolation, reverse transcription and first-stage PCR we hav e reduced the number of manipulations, maintained sensitivity, and minimize d the risk of contamination, A nested primer strategy is used to secure sen sitivity. We also introduce a competitive one-tube RT-PCR to be able to mon itor the relative quantity of transcripts using in vitro transcribed RNA as competitor.