Myeloid Leukemia

172 van der Reijden and Jansen
CBFB-MYH11 fusion transcripts have been reported to date. We discuss here
three CBFB-MYH11 qPCRs that allow for the detection of the most frequently
occurring fusion transcript types—A, D, and E. Together, these transcripts occur
in about 98% of all CBFB-MYH11–positive cases (see Table 2). The only
difference between these PCRs is the application of different primers located
in CBFB and MYH11 (see Subheading 3.3.1.). The CBFB-MYH11 real-time
probe described here intercalates with the minor groove of DNA (minor groove
binding [MGB] probe). Because these MGB probes bind DNA more efficiently
than standard real-time PCR probes, shorter sequences that exhibit similar
melting temperatures as compared to longer, non-MGB real-time PCR probes
can be used. In addition, these probes harbor a non-fluorescent quencher. As a
result, these probes exhibit lower background fluorescence than do probes containing
fluorescent quenchers.
3.3.1. Quantification of CBFB-MYH11 Positive Disease by qPCR
1. Prepare the CBFB-MYH11 qPCR mix by pipetting in the following order for one
PCR reaction (see Notes 2 and 3): 28.50 µL H 2O, 6.0 µL 25 mM MgCl 2, 0.25 µL
dNTPs, 5.0 µL 10X PCR buffer A, 1.5 µL primer Cfor (Table 1), 1.5 µL primer
MrevA, 2.0 µL probe CproMGB (use 5 pmol/µL instead of 10 pmol/µL), and
0.25 µL Taq gold DNA polymerase (total volume of 45.0 µL). To detect fusion
transcript types D and E, use as forward primer ENF803 instead of Cfor and use
reverse primers ENR863 or ENR865 instead of MrevA to detect respectively
fusion transcript types D and E (Table 1, Fig. 1).
2. Mix the CBFB-MYH11 qPCR mix and add per reaction 5.0 µL of cDNA (50 ng).
3. Perform the CBFB-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 Ct values and given quantities of the
calibration series.
3.3.2. Validation of Results and Quantification of Minimal Residual
Disease
Check whether the required sensitivity is observed (see Note 8) and check
the quality of the CBFB-MYH11 and PBGD PCRs according to Note 9. Check
the cDNA quality according to Note 10.
Normalize the CBFB-MYH11 expression of unknown samples (as generated
by TaqMan software) for PCR and cDNA input variations by multiplying the
quantity with the correction factor 2 ∆cycle threshold(Ct) , in which ∆Ct = Ct PBGD
of a sample minus the average PBGD Ct (see Note 10 for details). After normalization,
a maximal variation of fourfold in the quantity between duplicate
samples is allowed.