Myeloid Leukemia

WT1 Expression in AML and MDS 203
19. TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA).
20. ABI prism 7700 Sequence Detection System (Applied Biosystems, Foster City,
CA) (see Note 2).
21. PCR II TOPO vector (Invitrogen, Groningen, The Netherlands).
22. QIAFILTER Plasmid MIDI kit (Qiagen, Courtaboeuf, France).
23. BamHI and HindIII restriction enzymes.
24. Solution for dilution of standard curve plasmids: Tris 10 mM, ethylenediamine
tetraacetic acid (EDTA) 1 mM (pH 8).
25. Escherichia coli 16S and 23S rRNA (Roche).
26. ABL-containing plasmid for generation of the control gene standard curve is
available from Ipsogen (Marseilles, France).
3. Methods
3.1. Cell Separation and RNA Extraction
1. Collect 3–4 mL of BM in sodium citrate or 10–20 mL of PB in EDTA (see Note
3).
2. Dilute the sample with NaCl 0.9% solution or PBS. For PB, the dilution volume/
volume is 1:1; for BM, the dilution is 1 vol of BM to 2 vol of NaCl (or PBS).
3. Using a 10- to 15-mL centrifuge tube, layer 6 mL of the diluted blood or marrow
over 3 mL Lympholyte-H.
4. Centrifuge the tubes for 30 min at 800g at room temperature.
5. Using a pipet, carefully remove the mononuclear cells from the interface and
transfer to a 2-mL of Eppendorf tube.
6. Add 1.5 mL of NaCl 0.9% solution and centrifuge for 1 min at 800g.
7. Discard the supernatant and add 1 mL of RNAwiz (see Note 4) and homogenize
using a 2.5-mL syringe with a 22-gauge needle (see Note 5).
8. Incubate the homogenate at room temperature for 5 min.
9. Add 250 µL of chloroform and shake vigorously for 5 min (see Note 6).
10. Centrifuge the mixture at 12,000g for 15 min at 4°C.
11. Without disturbing the interphase, carefully transfer the aqueous phase into a 2mL
Eppendorf tube.
12. Add a half volume of sterile water and an equal volume of isopropanol and mix
well.
13. Store the tube at –20°C for at least 2 h (or overnight).
14. Centrifuge the tube for 15 min at 12,000g at 4°C.
15. Decant the supernatant and wash the pellet with 1 mL of cold 75% ethanol.
16. Centrifuge at 7500g for 5 min at 4°C.
17. Discard the supernatant and dry the pellet using a vacuum pump.
18. Resuspend the RNA in an appropriate amount of RNase-free water.
19. Assess the RNA concentration and purity by spectrophotometric analysis (at 260
nm and 280 nm).