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

Abstract

We describe a one-tube multiplex reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of bcr-abl fusion mRNA in analysis of patients with chronic myeloid leukaemia and acute lymphoblastic leukaemia. The assay provides a quick and reliable method for the detection and analysis of chromosome translocations resulting in formation of the fusion proteins p210 (b3a2/b2a2) and p190 (e1a2). The method is based on the use of magnetic beads and sequence-specific reverse transcription primers. By combining direct mRNA isolation, reverse transcription and first-stage PCR we have reduced the number of manipulations, maintained sensitivity, and minimized the risk of contamination. A nested primer strategy is used to secure sensitivity. We also introduce a competitive one-tube RT-PCR to be able to monitor the relative quantity of transcripts using in vitro transcribed RNA as competitor.