Myeloid Leukemia

Diagnosis of AML1-MTG8 (ETO)-Positive AML 151
1. 250 µL of MNCs are mixed with 400 µL of modified solution D. This should
give a final concentration of approx 4 M guanidinium isothiocyanate. Lysates
can be stored in solution D for prolonged periods at –80°C.
2. 65 µL of 2 M sodium acetate, pH 4.0, are then added to the sample and vortexed
well (see Note 1). Lysates can also be stored after the addition of sodium acetate.
3. Following 2–6 h incubation at room temperature, 650 µL of water-saturated phenol
are added and vortexed well.
4. 130 µL of chloroform are then added, the mixture is vortexed well, and incubated
on ice for 15 min.
5. Following centrifugation for 20 min at 13,000 rpm in a microfuge, the aqueous
layer is removed to a new tube, ensuring that the interface is not disturbed.
6. An equal volume of isopropanol is added to the aqueous layer, vortexed well, and
stored at –80°C for at least 2 h.
7. Following centrifugation for 15 min at 13,000 rpm, the supernatant is decanted
and the pellets are washed in 70% ethanol.
8. RNA pellets are then dried and re-suspended in sterile water (see Note 2).
9. RNA should be stored at –80°C or used for the RT reaction immediately.
3.2. Reverse Transcription
RT reactions are performed with random hexamer primers in a 20-µL reaction.
RT mixture is prepared by mixing the following on ice (see Note 3):
1. 4 µL of 5X first-strand buffer, 2 µL DTT, 0.8 µL dNTPs (mixture of all four dNTPs
at 25 mM concentration), 0.25 µL pdN(6) (at a concentration of 1 µg/µL), 1 µL
RNA Guard, 1 µL M-MLV reverse transcriptase, 5.95 µL water (see Note 4).
2. 2–4 µg of total RNA in a volume of 5 µL is heat denatured for 5 min at 75°C, then
snap cooled on ice for 3 min (see Note 5).
3. RNA is centrifuged, and 15 µL of RT mixture is added.
4. The reaction is performed by incubation at room temperature for 10 min, then at
37°C for 10 min, 42°C for 1 h, and 75°C for 5 min (see Note 6).
3.3. Qualitative RT-PCR
A number of protocols have been developed for the qualitative detection of
AML1-MTG8 (ETO). The sensitivity of these protocols varies from 10 –4 to 10 –6 .
This varied sensitivity could account for the conflicting data published using
these protocols. Using relatively sensitive RT-PCR protocols (10 –5 –10 –6 ), several
groups have shown that, following chemotherapy, autologous and allogeneic
bone marrow transplantation, AML1-MTG8 (ETO) transcripts can be
detected in most patients in long-term remission (6–8). However, other reports
have shown that negative qualitative RT-PCR results correlate with long-term
remission and cure (9).
For qualitative RT-PCR protocols, suitable controls are necessary. They
should include: