Real-time Quantification of BCR-ABL mRNA Transcripts Using the LightCycler-t(9;22) Quantification Kit

Literature indicates that in 95 % of all subjects with chronic myeloid leukemia (CML) and in 25-30 % of subjects with acute lymphoblastic leukemia (ALL) a reciprocal translocation between the long arms of chromosome 9 and chromosome 22 [t(9;22)] can be found. This translocation or the resulting fusion product can be detected by a number of techniques, including fluorescent in situ hybridization, Southern blotting, western blotting and reverse transcriptase polymerase chain reaction (RT-PCR). Of these techniques, RT-PCR for the chimeric fusion transcript BCRABL has received most attention in relation to the detection of minimal residual disease because of its high sensitivity (1). Attempts to research the remission and relapse events, occurring in the body of persons with CML after treatment with interferon-a or after bone marrow transplantation, requires sensitive techniques that detect smallest amounts of residual leukemic cells. Research concerning a possible correlation between a positive nested RT-PCR signal and the progression of the leukemia is hampered by the nonquantitative nature of conventional nested RT-PCR. Quantitative RT-PCR approaches that allow quantification of the transcript by competitive RT-PCR have been developed (2,3). The technique is based on serial dilutions of competitor contructs added to a constant amount of sample cDNA. Quantification is achieved by comparison of the relative band intensities of target and competitor PCR product on agarose gel. However, this method is very laborious since several concentrations of the competitor have to be used to cover the expected concentration range. In this article we present the LightCycler-t(9;22) Quantification Kit which is a research tool designed for the further study of possible relationships between the level of BCR-ABL fusion transcripts expression, stage of the disease, and response to treatment. The LightCycler System approach for the quantitative detection of the BCR-ABL fusion transcripts is based on real-time detection. The wide dynamic range of real-time PCR enables simultaneous analysis of samples with highly different starting concentrations, so that the initial target concentration can be determined in a single PCR. The concentration of BCR-ABL transcripts is determined relative to the number of transcripts of a control gene, glucose-6-phosphate dehydrogenase (G6PDH). The entire procedure from sample preparation to quantitative result is performed in 4.5 hours.