Myeloid Leukemia

150 Tobal and Yin
2. Materials
1. Ficoll-Hypaque, Amersham Pharmacia.
2. Modified solution D: 6 M guanidinium isothiocyanate, 42.5 M sodium citrate
(pH 7.0), 7.5% sarkosyl. Store in a dark container at 4°C.
3. Moloney murine leukemia virus (M-MLV) reverse transcriptase.
4. 5X first-strand buffer (supplied with M-MLV reverse transcriptase).
5. 2 µL dithiothreitol (DTT).
6. 25 mM dNTP mix.
7. Random hexamers, pdN(6).
8. Polymerase chain reaction (PCR) buffer (supplied with Taq polymerase).
9. W1 (Invitrogen, optional).
10. 50 mM MgCl 2.
11. Taq DNA polymerase.
12. Real-time qPCR master mix (2X), Eurogentec.
13. Phenol.
14. 70% ethanol.
15. Isopropanol.
16. Sterile water.
17. Primers:
a. Qualitative reverse-transcription (RT)-PCR (50 pmole/µL)
11—5� AGC CAT GAA GAA CCA GG 3� (first-round qualitative PCR)
12—5� AGG CTG TAG GAG AAT GG 3� (first-round qualitative PCR)
TS—5� CCC CGA GAA CCT CGA AAT CGT 3� (second-round qualitative PCR)
24—5� GTT GTC GGT GTA AAT GAA 3� (second-round qualitative PCR)
A2—5� TTC AGC GGC CAG TAG CAT CTG ACT T 3� (ABL qualitative PCR)
CA3—5� TGT TGA CTG GCG TGA TGT AGT TGC TTG G 3� (ABL
qualitative PCR)
b. Real-time quantitative RT-PCR (RQ-PCR) primers and probes (10 pmole/µL)
ABL forward: 5� TGTGGCCAGTGGAGATAACACT 3�
ABL reverse: 5� CATTCCCCATTGTGATTATAGCC 3�
ABL probe: 5� TAAGCATAACTAAAGGTGAAAAGCTCCGGGTCTT 3�
AML1-MTG8 forward : 5� CACCTACCACAGAGCCATCAAA 3� (see ref. 4)
AML1-MTG8 reverse: 5� ATCCACAGGTGAGTCTGGCATT 3�
AML1-MTG8 probe: 5� AACCTCGAAATCGTACTGAGAAGCACTCCA 3�
3. Methods
3.1. Sample Preparation and RNA Extraction
Mononuclear cells are obtained from peripheral blood (PB) or bone marrow
(BM) samples by Ficoll-Hypaque density gradient centrifugation, and should
either be stored at –80°C or used immediately for RNA extraction.
RNA is extracted from mononuclear cells (MNCs) by standard protocols.
The following protocol is a minor modification of the one published by
Chomczynski and Sacchi (5).