biological source: homo sapiens, childhood ALL patient (male, aged 5 years), peripheral blood taken after 24 hours of treatment (BFM2000 treatment protocol), leukocyte isolation without enrichment for malignant blasts. Technical protocols: Target hybridization preparation: For total RNA isolation, TRIreagent (MRC Inc., Cincinnati, OH,USA) was used according to the manufacturer’s protocol. Briefly, up to 1x10E7 cells per ml were lysed with TRIreagent, 200ul of chloroform were added, the mixture centrifuged, and the RNA from the aqueous phase precipitated with isopropanol. The pelleted RNA was washed in 70% ethanol in DEPC-water and resuspended in nuclease-free water. RNA quantity and purity was determined by optical densitiy measurements (OD260/280) and RNA integrity by using the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA,USA), and only high quality RNA was further processed. For Affymetrix GeneChip analysis 1.5ug total RNA was processed to generate a biotinylated hybridization target using One Cycle cDNA Synthesis and One Cycle Target Labelling kits from Affymetrix (Affymetrix, Santa Clara, CA, USA). All procedures were performed according to the manufacturer’s protocol (Gene Expression Analysis, Technical Manual, Revison 4; Eukaryotic Target Preparation, Revison 3) In brief, total RNA was reverse transcribed into cDNA using an anchored oligo-dT-T7-Primer, converted into double-stranded cDNA and purified with the Affymetrix Sample Clean-up Kit according to the manufacturer’s protocol. Thereafter, cRNA was generated by a T7-in-vitro transcription step including a modified nucleotide for subsequent biotinylation. Following RNA purification, 20ug of cRNA were fragmented at 95C using the Affymetrix fragmentation buffer, mixed with 200ul hybridization buffer containing hybridization controls (as contained in the Affymetrix One Cycle Target Labelling kit) and hybridized to U133 Plus 2.0 chips.
Hybridization: Measurement data /specifications: The chips were stained and washed in an Affymetrix fluidic station 450 following the EukGE-ws2v4 protocol. Fluorescence signals were recorded by an Affymetrix scanner 3000 and image analysis performed with the GCOS software (version 1.2).
Data processing: Data processing and analysis was performed in R using Bioconductor version 1.5. GeneChip raw expression values were normalized and summarized using the robust multiarray analysis (RMA) method proposed by R. Irizarry.
