Diagnosis and monitoring of chronic myeloid leukemia by qualitative and quantitative RT-PCR

Abstract

Real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR) methods for the quantitation of BCR-ABL mRNA in the blood of patients with chronic myeloid leukemia (CML) has become the predominant molecular monitoring technique. The BCR-ABL fusion gene is expressed in over 95% of patients with CML, and RQ-PCR provides a reliable, high-throughput method to accurately assess the level of treatment response and provides an early indication of emerging drug resistance. The ABI Prism 7700 Sequence Detection System uses TaqMan fluorogenic probes to quantitate specific nucleic acid sequences using RQ-PCR. The analyzer monitors an increase in fluorescence during the PCR cycle, which is proportional to the amount of accumulated product. The starting copy number is calculated relative to a series of standards. The copy number is normalized to a control gene that compensates for variations in the efficiency of the RT step and for the degree of RNA degradation. In our experience, reliable and consistent RQ-PCR requires thorough validation of all aspects of the procedure, including the selection of an appropriate control gene, careful assay design to avoid polymorphisms in primer or probe binding sites and to exclude the amplification of contaminating DNA, and monitoring the performance of the RQ-PCR by the use of quality control samples.