Myeloid Leukemia

4 Jansen and van der Reijden
2. Materials
1. Diethylpyrocarbonate (DEPC).
2. Guadinine thiocyanate (GTC) solution: 4 M guanidinium thiocyanate, 0.5% Nlaurolylsarcosine,
25 mM sodium citrate (pH 7.0), 0.1 M 2-mercaptoethanol
(added shortly before use).
3. CsCl solution for RNA ultracentrifugation: 5.7 M CsCl in 0.1 M sodium-ethylenediamine
tetraacetic acid (EDTA) (pH 8.0). Treat with DEPC and autoclave.
4. DEPC-treated H 2O.
5. Ethanol.
6. Agarose.
7. Reverse transcriptase.
8. 0.1 M Diothiothreitol (DTT).
9. dNTPs, 25 mM each.
10. Random hexamers, 5 mg/mL.
11. RNasin ribonuclease (RNase) inhibitor.
12. 10X TBE buffer: 108 g Tris-base, 55 g boric acid, 40 mL 0.5 M EDTA (pH 8.0);
add H 2O to 1 L.
13. Sodium dodecyl sulphate (SDS).
14. 6X loading buffer containing SDS: 0.25 g bromphenol blue, 0.25 g xylene cyanol,
and 15 g Ficoll in 100 mL H 2O; add 10% SDS w/v.
15. Ethidium bromide.
16. SE buffer: 12.5 mL 100 mM EDTA plus 3.75 mL 1 M NaCl in 50 mL autoclaved
H 2O.
17. Proteinase K, 20 mg/mL.
18. Saturated NaCl: 87.7 g NaCl in 250 mL.
19. TE buffer: 10 mM Tris-HCl, 1 mM EDTA (pH 8.0).
20. Microcentrifuge.
21. Ultracentrifuge.
22. Spectrophotometer.
23. Superscript II reverse transcriptase RNase H - , and buffer (Invitrogen).
24. Moloney murine leukemia virus (M-MLV) reverse transcriptase and buffer
(Invitrogen).
3. Methods
In this section, the isolation of RNA and DNA, and the synthesis of cDNA
are described.
3.1. Isolation of RNA
For the detection of most fusion genes and several other mutations that are
recurrently found in hematological malignancies, total RNA can be used. Both
bone marrow and peripheral blood nucleated cells may be used, depending on
the question asked and the stage of the disease (see also Introduction). EDTA
or citrate are preferred as anticoagulant (see Note 1). Various protocols to iso-