Myeloid Leukemia

156 Tobal and Yin
Fig. 3. The TaqMan 5� nuclease assay.
2. AML1-ETO RQ reaction mix: in a 25-µL reaction volume, mix 12.5 µL qPCR
master mix, 1 µL t(8;21)-forward primer, 1 µL t(8;21)-reverse primer, 0.5 µL
t(8;21)-probe, 5 µL H 2O.
3. Reaction mixes for both ABL and AML1-ETO RQ should be prepared in sufficient
amounts for the number of reactions needed.
4. 20 µL of reaction mix is added in each well.
5. 5 µL of each cDNA (see Note 11) is added in triplicate wells for each sample.
Standard curves should be performed once a month using constructs prepared in
the laboratory or commercially available ones (see Note 12). Figure 4 shows
examples of standard curves for ABL and AML1-MTG8 (ETO) transcripts.
6. Negative controls (reactions without cDNA) should be included in every plate.
7. Wells should be sealed. The plate is then centrifuged at 3000 rpm for 3 min.
8. Real-time amplification is then performed according to the following parameters:
50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and
60°C for 1 min.
9. Analyze the data.
10. Levels should be expressed as a ratio of AML1-MTG8 (ETO)/ABL transcripts.