An example of a real time quantitative fluorescence RT-PCR assay (Q-RT-PCR) for detection of minimal residual disease (MRD) in chronic myelogenous leukemia (CML).
(A) The graph shows a typical standard curve, generated by Q-RT-PCR of a series of serial dilutions of a BCR/ABL (+) cell line. BCR/ABL transcripts in RNA from clinical samples are quantitated by comparison to this standard curve. p210 and p190 BCR/ABL transcripts, in both cell line standards and patient samples, are normalized to G6PDH housekeeping gene. The fluorescence signal generated during the PCR process is proportional to the starting quantity of BCR/ABL template, and is detected by a fluorimeter in the instrument. The PCR reaction is monitored in real time, during the geometric phase of the reaction; data from the linear and plateau phases are not used. The standard test will detect 1 BCR/ABL (+) cell in 105 normal cells. (B) Increased analytical sensitivity of 1 (+) cell in 106-7 normal cells can be obtained by concentration of sample RNA used in the analysis.
