ATP was purchased from Sigma-Aldrich (Milan, Italy). Blast cells obtained from 12 AML patients were cultured for 24 h with or without 1 mM ATP.
Growth protocol
Primary leukemic cells were obtained from the peripheral blood (PB) of 12 AML patients at diagnosis, before treatment. Mononuclear cells (MNC) were separated from AML samples, immediately after peripheral blood harvesting by Ficoll-Hypaque centrifugation (Amersham Bioscience, Piscataway, NJ, USA). Blast cells obtained from AML patients were cultured in IMDM + 10%FBS.
Extracted molecule
total RNA
Extraction protocol
Total RNA from 106 ATP-treated and untreated cells was extracted using RNeasy Micro Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s recommendations. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit; Agilent Technologies, Waldbrunn, Germany) were used to determine the purity/integrity of RNA samples using Agilent 2100 Bioanalyzer. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to evaluate the RNA sample concentration, and 260/280 and 260/230 nm ratios were considered to verify the purity of total RNA.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard One Cycle Affymetrix protocol from 1 ug total RNA (Gene Chip Expression Analysis Technical Manual, 701024 Rev3, Affymetrix). Labeled cRNA was purified using Affymetrix GeneChip Sample Cleanup Module and fragmented (15 µg) as described in the Affymetrix GeneChip protocol. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) and Agilent 2100 Bioanalyzer were used to determine the cRNA concentration/quality as well as to optimize the cRNA fragmentation.
Hybridization protocol
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on Affymetrix HG-U133 Plus2 GeneChip arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 using the instrument’s standard FS450_0001 protocol with antibody-mediated signal amplification and GeneChip Hybridization, Wash and Staining Kit.
Scan protocol
GeneChip were finally scanned using the Affymetrix GeneChip scanner 3000, enabled for High-Resolution Scanning.
Description
Gene expression data from untreated AML blasts
Data processing
The GeneChip-Operating-Software (GCOS) absolute analysis algorithm was used to determine the mRNA expression levels (Signal), while the GCOS comparison analysis algorithm was used in order to compare gene expression levels between two samples. The data were analyzed with GCOS using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 150.
