Myeloid Leukemia

214 Kohlmann et al.
2. Materials
A detailed description of reagents and required buffers is also available in
the Affymetrix technical manual on gene expression profiling analyses
(www.affymetrix.com).
1. Safe-Lock tubes (Eppendorf).
2. Acetylated bovine serum albumin (BSA), 50 mg/mL (Sigma).
3. Ammonium acetate, 7.5 M (Sigma).
4. cDNA Synthesis System Kit (Roche Applied Science).
5. Control oligonucleotide B2, 3 nM (Affymetrix).
6. Ethylenediamine tetraacetic acid (EDTA), 0.5 M (Sigma).
7. Enzo BioArray HighYield RNA Transcript Labeling Kit (Affymetrix).
8. Ethanol (Roth).
9. Eukaryotic Hybridization Control Kit, 20X stock solution (Affymetrix).
10. Ficoll Isotonic Solution (density: 1077 g/mL) Biochrom (Berlin).
11. Fragmentation buffer, 5X concentrated (Affymetrix).
12. Glycogen, 20 mg/mL (Roche Applied Science).
13. Guanidine isothiocyanate buffer (RLT-Buffer, Qiagen).
14. Herring sperm DNA, 10 mg/mL (Sigma).
15. Hybridization buffer, 2X concentrated.
16. Nuclease-free water (Ambion).
17. Phosphate-buffered saline (PBS) with Ca 2+ and Mg 2+ .
18. Phase Lock Gel light (Eppendorf).
19. Phenol/chloroform/isoamylalcohol, 25:24:1 (Ambion).
20. QIAshredder columns (Qiagen).
21. RNeasy Mini Kit (Qiagen).
22. Streptavidin-phycoerythrin (SAPE) staining solution.
3. Methods
3.1. Patient Samples
All leukemia patient samples should be sent to the laboratory via express
mail. At the time point of diagnosis, the patients should provide bone marrow
(BM) aspirates or peripheral blood samples. All relevant clinical parameters,
as well as detailed diagnostic reports, can be entered in a specific leukemia
database (1).
3.2. Isolation of Mononucleated Cells by Ficoll Gradient Centrifugation
1. Mix 1–20 mL of BM or blood with the same volume of PBS.
2. Place the same volume as this total mixture (or at most 20 mL) of Ficoll into an
extra tube. Carefully overlay the Ficoll with the BM/PBS mixture and centrifuge
for 20 min at 1088g. Don’t use the brake; it will last around another 20 min until
the run stops.