LightCycler technology for the quantitation of bcr abl fusion transcripts

Quantifying bcr/abl fusion transcripts in chronic myelogenous leukemia is t hought to serve as a powerful parameter for monitoring the kinetic nature o f this clonal disease in vivo and in vitro. Recently, we demonstrated the t echnical advantages as well as the clinical relevance of quantitating bcr/a bl fusion mRNA using the 5-nuclease assay and a real-time fluorescence reve rse transcriptase-PCR (RT-PCR) detection system (ABI PRISM 7700 SDS). Meanw hile, another technique was introduced (LightCycler technology) that may be used for the same purpose. To investigate whether this method may be an ap propriate alternative to the described procedure, we have established bcr/a bl LightCycler RT-PCR for major and minor bcr/abl fusion transcripts. We fo und that, with only minor modifications, TaqMan RT-PCR and fluorescent prob e design can be used to obtain comparable results in the LightCycler system . The developed method could quantitate as little as 10 bcr/abl copies per 100 ng cDNA and was as safe and reproducible as the previously described te chnique. Because reaction efficiency was identical within different bcr/abl major fusions, one single RT-PCR could be established that simultaneously detects b2a3, b2a2, b3a2, and b3a3 fusion RNA with equal specificity and se nsitivity. Compared to results generated by the ABI PRISM 7700 SDS, absolut e amounts of bcr/abl did not differ significantly, and there was a linear c orrelation between the respective values. We conclude that TaqMan chemistry can be used in the LightCycler and that both real-time fluorescence PCR de tection systems equally fulfill the criteria for the safe and reliable quan titation of bcr/abl fusion RNA in clinical samples. This may be of help for further standardization of quantitative bcr/abl RT-PCR, which, again, is n ecessary for the comparison of results generated by different investigators .