Myeloid Leukemia

152 Tobal and Yin
1. A negative (no-reaction) control.
2. A negative sample control (a sample or cell line negative for t(8;21)).
3. A positive control (a sample or cell line positive for t(8;21)).
4. A control gene, such as ABL, performed on all samples.
Samples must be tested for a suitable control gene. We have found the ABL
gene to be suitable for most of the leukemia fusion genes.
3.3.1. ABL PCR Amplification
1. For ABL detection, the PCR reaction is performed in a 25-µL volume containing:
2.5 µL PCR buffer, 1.25 µL W1 (optional), 0.75 µL MgCl 2 (50 mM), 0.25
µL dNTPs (25 mM), 0.25 µL each primer (A2, CA3), 0.3 µL Taq DNA polymerase,
17.45 µL H 2O (see Note 7).
2. 2 µL of cDNA is added to the PCR mixture, vortexed, and centrifuged briefly.
3. PCR amplification is then performed according to these parameters:
a. 95°C for 3 min
b. 40 cycles of 93°C × 1 min, 55°C × 1 min, 72°C × 1 min.
c. 72°C for 5 min.
4. 10 µL of PCR products should be electrophoresed on a 2% agarose gel. Expected
band size is 276 bp (see Notes 8 and 9).
3.3.2. AML1-MTG8 (ETO) PCR Amplification
Once ABL amplification is successful, the sample is ready for AML1-MTG8
(ETO) amplification. The protocol described as follows has a sensitivity of 10 –6 .
The protocol takes approx 10 h (two rounds of 4–5 h, depending on the PCR
machine used).
1. PCR reaction is performed in a 50-µL volume (see Note 7).
2. First-round PCR master mix is prepared as follows: 5 µL PCR buffer, 2.5 µL W1
(optional), 1.5 µL MgCl 2 (50 mM), 0.5 µL dNTPs (25 mM), 0.25 µL each of
primer 11 and primer 12, 0.3 µL Taq DNA polymerase, 37.7 µL H 2O.
3. 2 µL of cDNA is added to the PCR mixture, vortexed, and centrifuged briefly.
4. PCR amplification is then performed according to these parameters: 95°C for 3
min, then 40 cycles of 93°C 1 min, 55°C 1 min, 72°C 1 min, followed by 72°C
for 5 min.
5. A second round PCR amplification of AML1-MTG8 (ETO) is then performed on
2 µL first-round product, using primers TS and 24. PCR parameters are the same
as those used for the first-round PCR.
6. 10 µL of PCR products should be electrophoresed on a 2% agarose gel. Expected
band size is 152 bp in a positive sample (see Note 9).
3.4. Minimal Residual Disease (MRD) Monitoring
The main aim of MRD monitoring is to assess the effectiveness of treatment
and to predict relapse at an early stage, thus possibly allowing preemptive or