Myeloid Leukemia

164 van der Reijden and Jansen
tify minimal residual disease during and after treatment with real-time quantitative
CBFB-MYH11 reverse-transcription (RT)-polymerase chain reaction
(PCR). Because the sensitivity of real-time RT-PCR allows the quantification
of at least 1 malignant cell in 10,000 normal cells, this technique allows the
early identification of patients who relapse (3,4).
2. Materials
1. cDNA synthesized from RNA isolated from peripheral blood or bone marrow
cells.
2. Oligonucleotide primers, final concentration 10 pmol/µL (see Table 1).
3. Real-time PCR probes, final concentration 10 pmol/µL (see Table 1).
4. A 25 mM dNTP solution. This solution is prepared by mixing equal amounts of
100 mM dATP, dCTP, dGTP, and dTTP.
5. 10X PCR buffer.
6. MgCl2. 7. Taq DNA polymerase.
8. Dimethylsulfoxide (DMSO).
9. Mineral oil.
10. TAQ gold DNA polymerase (Applied Biosystems).
11. Agarose gel and DNA sequencing equipment.
12. 6X DNA loading buffer containing 5% sodium dodecyl sufate (SDS).
13. Classical PCR equipment.
14. Real-time PCR equipment (Taqman PCR 7700 or 7900, Applied Biosystems).
For storage conditions, see Note 1.
3. Methods
The methods in this chapter outline (1) the detection of the CBFB-MYH11
fusion gene at diagnosis by qualitative and quantitative RT-PCR and (2) the
quantification of CBFB-MYH11-positive cells during and after treatment by
quantitative RT-PCR. Because the genomic breakpoints in CBFB and MYH11
are scattered over large areas, it is not feasible to amplify these by regular
PCR. However, at the RNA transcript level the breakpoints are found within a
limited region. Therefore, reverse transcriptase PCR is used for the detection
of the CBFB-MYH11 fusion. To this end, RNA isolated from peripheral blood
or bone marrow cells is first subjected to cDNA synthesis using reverse transcriptase.
The synthesized cDNA is subsequently used as the target in CBFB-
MYH11 PCR.
3.1. Diagnosis of CBFB-MYH11-Positive Acute Myeloid Leukemia
by Qualitative CBFB-MYH11 RT-PCR
To date, at least 10 different in-frame CBFB-MYH11 fusion transcripts have
been reported, which are caused by variable breakpoints in both CBFB and