Leukemic cells were isolated and enriched from these samples. Within 24 hours after sampling, mononuclear cells were isolated by density gradient centrifugation using Lymphoprep (density 1.077 g/mL; Nycomed Pharma, Oslo, Norway), and centrifuged at 480g for 15 minutes at room temperature. The collected mononuclear cells were washed twice and kept in culture medium consisting of RPMI 1640 medium (Dutch modification without L-glutamine; Gibco BRL, Life Technologies, Breda, The Netherlands), 20% fetal calf serum (FCS; Integro, Zaandam, The Netherlands), 2 mM L-glutamine (Gibco BRL, Life Technologies) 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL sodium selenite (ITS media supplement; Sigma, St Louis, MO), 100 IU/mL penicillin, 100 µg/mL streptomycin, 0.125 µg/mL fungizone (Gibco BRL, Life Technologies), and 0.2 mg/mL gentamycin (Gibco BRL, Life Technologies). Contaminating nonleukemic cells were removed by immunomagnetic beads. as described by Kaspers et al (1994). All resulting samples contained 90% leukemic cells, as determined morphologically by May-Grünwald-Giemsa-stained cytospins (Merck, Darmstadt, Germany). Viably frozen T-ALL cells were used for DNA and RNA extraction, and a minimum of 5 million leukemic cells were lysed in Trizol reagent (Invitrogen, Life Technologies, Breda, The Netherlands) and stored at -80°C.
Extracted molecule
total RNA
Extraction protocol
Genomic DNA and total cellular RNA were isolated using Trizol (Invitrogen) according to the manufacturers’ protocol, with minor modifications. An additional phenol-chloroform extraction was performed and the RNA was precipitated with isopropanol along with 1 mL (20 mg/mL) glycogen (Roche, Almere, The Netherlands). After precipitation, RNA pellets were dissolved in 20 mL RNAse-free TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH=8.0). The RNA concentration was quantified spectrophotometrically. Integrity of total RNA was checked using the Agilent 2100 Bio-analyzer (Agilent, Santa-Clara, USA).
Label
biotin
Label protocol
Copy-DNA and ccRNA syntheses from total RNA, hybridization of Humane Genome U133 plus2.0 oligonucleotide microarrays (Affymetrix, Santa-Clara, USA) and washing steps were performed according to the manufacturers’ protocol.
Hybridization protocol
Hybridization of Humane Genome U133 plus2.0 oligonucleotide microarrays (Affymetrix, Santa-Clara, USA) and washing steps were performed according to the manufacturers’ protocol.
Scan protocol
GeneChips were scanned using the Affymetrix Genechip scanner 3000
Description
pediatric T-ALL patient #63
Data processing
Probeset intensities were extracted from CEL-files using GeneChip Operating Software (GCOS), version 1.4.0.036 (Affymetrix). Probe intensities were normalized using the variance stabilization procedure (Bioconductor package VSN) in the statistical data analysis environment R, version 2.2.0. Differentially expressed genes between T-ALL subgroups were calculated using a Wilcoxon statistical test, and corrected for multiple testing error according to the false discovery rate procedure as developed by Hochberg and Benjaminin using the Bioconductor package Multtest.
