Myeloid Leukemia

Quantitative PCR for Monitoring CML Patients 71
Fig. 1. Amplification plot and standard curve generated by real-time quantitative
reverse-transcription polymerase chain reaction of BCR-ABL. Ten-fold serial dilutions
of plasmids for each gene are analyzed in every real-time quantitative reverse-transcription
polymerase chain reaction (RQ-PCR) analysis to generate a standard curve
for quantitative analysis. (A) PCR amplification plots of the 10-fold serial dilutions of
the b3a2 BCR-ABL standard with known starting copy number of approx 10 6 to 10
copies. The accumulation of fluorescence occurs at each cycle. The 7700 software
calculates the fractional cycle number (cycle threshold [Ct]) where the amplification
plot crosses a defined fluorescence threshold. The Ct values are used to generate a
standard curve (B). The correlation coefficient should be between 0.997 and 1.0, and
the slope value for the plasmid standards lies between –3.3 and –3.59. The Ct values of
the unknown samples are calculated from the standard curve.