Primary CLL samples were cultivated using RPMI 1640 (Biochrom AG, Berlin, Germany), supplemented with 10% FCS (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-Glutamin (Biochrom AG), and Penicillin/Streptomycin (GIBCO, Invitrogen Corporation, Grand Island, NY, USA). Prior to treatment, cells were stained with trypan blue (Sigma-Aldrich), counted, and diluted to a density of 1.0x106 cells/ml. Thawing of viably frozen samples followed the DSMZ (German Collection of microorganisms and cell lines, Braunschweig) guideline. Agents used to treat primary AML sample for 4 and 20 hours in vitro were DMSO (control/carrier; dimethyl sulfoxide; Sigma-Aldrich), and BV6 was synthesized at Genentech, Inc. (South San Francisco, CA, USA).
Extracted molecule
total RNA
Extraction protocol
The total RNA was purified with a TRIzol-based protocol.
Label
Biotin
Label protocol
For each sample preparation, total RNA was converted into double-stranded cDNA by reverse transcription using the 3' IVT Express Kit from Affymetrix according to the manufacturer's recommendations.
Hybridization protocol
Hybridization, washing, and staining protocols, respectively, were performed on Affymetrix GeneChip instruments (Hybridization Oven 640, Fluidics Station FS450) as recommended by the manufacturer.
Scan protocol
Scanning was performed on Affymetrix GeneChip Scanner GCS3000 instruments as recommended by the manufacturer using default settings. The software used was GCOS 1.2 or higher.
Description
50_20h_BV6
Data processing
The data were analyzed with BRB ArrayTools using RMA. Intensity values were log2 transformed
