Myeloid Leukemia

170 van der Reijden and Jansen
determine the type of CBFB-MYH11 fusion transcript. The type of CBFB-
MYH11 transcript can be determined with qualitative CBFB-MYH11 PCR followed
by sequence analysis of the amplified PCR product.
Because cryopreservation can negatively affect the percentage of CBFB-
MYH11-positive cells, quantitative MYH11 PCR is suitable only as an indicator
of the presence of CBFB-MYH11 in freshly isolated samples.
To enable quantification of MYH11 expression in patient samples, a calibration
series with known amounts of input covering the desired range of quantification
must be included in each PCR. The best and most convenient way to
show that the desired sensitivity is achieved is to use a complete dilution series,
consisting of cDNA generated from a positive control (e.g., the cell line ME-1
[11]) diluted into H 2O. Such a dilution series should be included in each analysis.
1. Prepare the MYH11 qPCR mix by pipetting in the following order for one PCR
reaction (see Notes 2 and 3): 24.25 µL H 2O, 10.0 µL 25 mM MgCl 2, 0.5 µL
dNTPs, 5.0 µL 10X PCR buffer A, 1.5 µL primer Mfor (Table 1), 1.5 µL primer
Mrev (Table 1), 2.0 µL probe Mpro, and 0.25 µL Taq gold DNA polymerase
(total volume of 45.0 µL).
2. Mix the MYH11 qPCR mix and add per reaction 5.0 µL of cDNA (50 ng).
3. Perform the MYH11 qPCR using an initial denaturing step of 10 min at 94°C to
activate the Taq gold DNA polymerase, followed by 45 cycles of 15 s at 94°C
(denaturation) and 1 min at 60°C (primer and probe annealing and extension).
4. Collect the quantities of the unknown samples generated by the real-time PCR
equipment that are based upon the obtained given quantities and generated Ct
values of the calibration series.
3.2.2. Quantitative PBGD PCR for Normalization
The quantification of a control gene should be performed on each patient
sample analyzed in real-time PCR to correct for variations in PCR and cDNA
synthesis efficiency, cDNA input variations, and so on. However, if values are
normalized over large ranges (>10-fold differences based upon the reference
gene), quantitative results may be quite imprecise. To avoid correction over
large ranges, we strongly recommend applying the same amount of cDNA in
each PCR. Another advantage of this approach is that a calibration curve for
quantification of the reference gene is not needed, because normalization occurs
only over a limited range. By following this strategy, the amplification
(quantification) of the control gene also shows that cDNA was efficiently synthesized.
Several control genes for real-time RT-PCR normalization have been
evaluated (14). We discuss here amplification of the PBGD (porphobilinogen
deaminase) gene, because the variation in expression in bone marrow and blood
samples is minimal (see Table 1 for PBGD primer-probe combination, and
Note 7).