Hybridization controls

Total RNA was extracted from flash frozen cells using the AllPrep DNA/RNA/protein Mini kit (Qiagen, Hilden, Germany), quality-controlled using a 2100 BioAnalyzer (Agilent, Santa Clara, CA, USA) and quantified using a Nanodrop 2000c spectrophotometer (Thermo Scientific, Wilmington, USA). Only samples with an RNA Integrity Number ‘RIN’ >8.0 were used for further gene expression analysis. Using the WT Expression Kit (Ambion Inc, Austin, TX, USA), cDNA was prepared from 10 μg of purified cRNA, originally synthesised and purified from 0.25 μg of total RNA following the manufacturer's instructions. The cDNA (2.75 μg) was then used for fragmentation and labeling using GeneChip Terminal Labelling Kit (Affymetrix, Santa Clara, CA, USA). Using GeneChip Hybridization, Wash and Stain (Hybridization module) (Affymetrix, Santa Clara, CA, USA), and Hybridization controls (Affymetrix, Santa Clara, CA, USA), fragmented and labelled cDNA was hybridized to Mouse Gene 2.0 ST Arrays (Affymetrix, Santa Clara, CA, USA). After hybridization under orbital rotation for 16 h at 45°C, arrays were washed and stained using GeneChip Hybridization, Wash and Stain Kit (Stain module) (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's instructions. Finally, arrays were scanned immediately using Affymetrix GeneChip Scanner GS 3000.

The quality of RNA obtained from each AML and HD lymphocyte sample was assessed based on the RNA integrity using a Bioanalyser 2100 (Agilent Inc., Diegem, Belgium). Total RNA (100 ng) was converted to double-stranded cDNA in a reverse transcription reaction. The obtained cDNA was converted and amplified to antisense cRNA and labeled with biotin in an in vitro transcription reaction using an HT 3′ IVT Affymetrix Express Kit. All steps were carried out according to the manufacturer’s protocol (Affymetrix) [25 (link)]. A mixture of purified and fragmented biotinylated cRNA and hybridization controls (Affymetrix) was hybridized on Affymetrix GeneChip arrays followed by staining and washing. To assess the raw probe signal intensities, chips were scanned using a Gene Chip scanner 3000 (Affymetrix). The GeneChip™ Human Genome U133 Plus 2.0 Array (Affymetrix), which analyzes over 47,000 transcripts, was used in the study. The GC-Robust MultiChip Average (GCRMA) [26 (link)] was used as part of the GCRMA package at the Bioconductor site (http://www.bioconductor.org) to preprocess the raw data (CEL files).