cell classification: aggressive leukemia cell type: chronic lymphocytic leukemia B-cell unstimulated (us)/stimulated (s): S time point (min): T90 transfected with: non-targeting RNAi
Treatment protocol
cell isolation: B-cells were negatively selected from fresh blood samples using the Rosettesep B-cell enrichment cocktail (Stemcell Biotechnologies, Vancouver, Canada) and isolated by density gradient centrifugation over Ficoll-Paque plus (Pharmacia, Upsala, Sweden). Quality of the selection was assessed by flow cytometry on a Cytomics FC500 system (Beckman-Coulter, Fullerton, CA) after CD5-PE / CD19-FITC staining (BD Biosciences, Palo Alto, CA) and in all cases was greater than 93%, range from 93-98% (med. 96%). Cells were starved for 4 hr at 37degree C / 5% CO2 in RPMI 1640 media supplemented with 2 nM L-glutamine and 24 ug/mL gentamicin. B-cells at a density of 10^7cells/mL were divided in two. One was used as unstimulated (US) control and the other stimulated (S) by goat F(ab’)2 anti human IgM-BIOT (Southern biotech, Birmingham, AL) at 20 mg/mL and cross-linked by 20 mg/mL of avidin (Sigma-Aldrich, St Louis, MO) on ice during 5 min, follow by incubation at 37degree C for 15 min. After washing, cells were cultured at 37degree C / 5% CO2 in RPMI 1640 media supplemented with 10% FBS, 2 nM L-glutamine and 24 ug/mL gentamicin.
Extracted molecule
total RNA
Extraction protocol
At each time point 10^7 control US and S cells were washed and resuspended in 1 mL TRIzol (Invitrogen, Carlsbad, CA). Total RNA was purified using Promega columns (SV total isolation system, Promega corp. Madison, WI).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from total RNA.
Hybridization protocol
standard affymetrix
Scan protocol
standard affymetrix
Description
CLL-UM4_Transfect-non target_S_T90
Data processing
Data were normalized with the invariant set method and the model based expression index (MBEI) obtained by the PM-MM model (Li et al., PNAS 2001) using dChip software (dChip v.2006; www.dchip.org). The obtained expression values are preprocessed by truncating the value to less than or equal to 1.
