Supplemental methods
Affymetrix microarray analysis
BM samples were collected at diagnosis, before drug treatment and following informed consent from patients who had enrolled in the MRC-UK AML clinical trial protocol (http://www.aml14.bham.ac.uk/ or http://www.aml15.bham.ac.uk/). Patient and healthy donor-derived blast cells were washed in PBS and high-quality total RNA was extracted by lysis in Trizol, according to the manufacturer’s instruction (Invitrogen, Paisley, U.K.). For culture cells, total RNA was extracted from GFP (control) and RUNX1-RUNX1T1 matched transduced human progenitor cells (which were >95% CD34+ and ~70% GFP+ on day three of culture). RNA from myeloblasts, monoblasts and erythroblasts (n=4 for each matched set on culture day 6 were also extracted. The high frequency of retroviral transduction in these samples made it unnecessary to purify GFP+ cells for this analysis. RNA quantity, quality, and purity were assessed with the use of the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technolgies, Stockport, U.K.). None of the samples showed RNA degradation (ratio of 28S ribosomal RNA to 18S ribosomal RNA of at least 1.8) or contamination by DNA.
Total RNA (7.5µg) from each sample was used to generate first-strand cDNA with a T7-Oligo(dT) promoter primer (Affymetrix, High Wycombe, U.K.). Following second-strand synthesis, in vitro transcription was performed using the BioArray, HighYield RNA transcript labelling kit (Enzo Life Sciences Inc., Farmingdale, NY) according to the manufacturer’s instructions. The preparation and processing of labelled and fragmented cRNA targets, as well as Affymetrix HG-U133A GeneChip hybridization and scanning was carried out according to the manufacturer’s protocol (http://www.affymetrix.com). The GeneChips (which contain 22,283 probe sets or genes, representing approximately 14,500 different genes), were scanned using a Hewlett Packard confocal laser scanner and analyzed using MicroArray Suite version 5.0(MAS) (Affymetrix) (see “Affymetrix microarray data analysis”). Overall, day 3 and day 6 transduced cells showed on average significant levels of present calls in the hybridisation arrays (50.2% ± 0.02 and 45.8% ± 0.08) demonstrating the high quality of Affymetrix data. The Affymetrix data is available as supplemental material on http://www.ebi.ac.uk/arrayexpress/query/entry (accession number a-mexp-583).
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