Myeloid Leukemia

Quantitative PCR for Monitoring CML Patients 85
4°C will rapidly degrade the BCR-ABL mRNA.
2. The selection of an appropriate control gene constitutes one of the most important
aspects for accurate RQ-PCR. For example, GAPDH is frequently used as a
control for RQ-PCR assays. However, there is overwhelming evidence suggesting
that its use is inappropriate (reviewed in refs. 3,4). The control should ideally
have a similar expression level to the target and degrade at a similar rate to compensate
for variations in the quality of the RNA between samples. We have chosen
the normal BCR gene, as it has a similar expression level to BCR-ABL and it
degrades at a similar rate (14). To compensate for differences in the efficiency of
each RT reaction, the control gene and the BCR-ABL gene are reverse transcribed
in the same reaction. Calculating a ratio of BCR-ABL to BCR compensates for the
variations.
3. The primers and probes were designed using Primer Express software and specifically
amplify RNA. One of the primers hybridizes at the junction of an exon
boundary. The amplicon sizes for the three transcripts are all less than 100 bp,
which allows a sensitive detection system. One K562 cell is detectable in a background
of 10 5 BCR-ABL-negative cells. During the development of the TaqMan
assay, we validated the BCR-ABL quantitative values by correlation with the percentage
of the Philadelphia chromosome as assessed by cytogenetic analysis. A
good correlation was found, except that a small percentage of patients with the
b2a2 BCR-ABL transcript had lower quantitative values than expected. By
sequence analysis of the PCR amplicon of these patients, it was found that they
all had a polymorphism located at the hybridization site of the sixth base from the
3� end of the reverse primer. The efficiency of primer annealing was reduced in
the patients with the polymorphism, which resulted in an approx 10-fold lower
quantitative value. The problem was overcome by redesigning the primer, which
avoided the polymorphic site (see Fig. 2). These data highlight the importance of
avoiding polymorphisms when designing probes and primers, although the position
of these sites is not always known, as in our case.
4. We routinely analyze the three separate PCR reactions that are required for the
quantitation of BCR-ABL on the same 96-well plate, which contains the three
different standard curves. The SDS software will analyze only one standard curve
per reaction, and therefore each set of data for the three transcripts must be saved
into a separate file for analysis. This is done by copying the original file into
three separate files and assigning all of the wells, except for those to be analyzed
for a particular transcript, as “not in use.” The three files are analyzed separately.
We set a threshold of 0.04 and a baseline reading between 3 and 15, which is
usually sufficient to ensure that the threshold is above the background fluorescence
readings and within the linear portion of the curves. The threshold may
need to be increased on the very rare occasion that the linear portion is not
reached. The standard curve is analyzed and accepted if the slope value is between
–3.3 and –3.59 and the correlation coefficient is between 0.997 and 1.0. If
any point on the standard curve is an obvious outlier it can be removed and the
standard curve reanalyzed. We have not found it necessary to run the standard