Kit225 cells at a density of 8x10E5/ml were treated with 25 mM NC1153 or 0.1 % DMSO for 12 hours.
Growth protocol
Kit225 cells were maintained in RPMI-1640 medium containing 10% fetal calf serum, 2 mM L-glutamine and penicillin-streptomycin supplemented with 20 U/ml human recombinant IL-2.
Extracted molecule
total RNA
Extraction protocol
QIAGEN RNeasy Kit. RNA samples were analyzed for purity (A260/280 min. 1.9, A260/230 min 1.7) and integrity (capillary electrophoresis for degradation and DNA contamination) on an Agilent 2100 Bioanalyser and the NanoDrop ND-1000 Spectrophotometer. Only RNA samples that passed this Quality Check were used for subsequent hybridization.
Label
biotin
Label protocol
Affymetrix Labeling (Total RNA). These steps as well as the QC steps were perfomed by MCF according to their standardized procedures.
Hybridization protocol
Affymetrix Hybridization, Washing/Staining on the Fluidics Station
Scan protocol
Affymetrix GeneChip® Scanner 3000
Description
12h treatment
Data processing
Array quality parameters were as follows: Scaling Factor (6.681, 2.881, 2.967, 2.656 for NC1153_1, DMSO_1, NC1153_2, DMSO_2, respectively), Average Background (65.71, 87.2, 72.77, 79.23), number of present probes for housekeeping probe sets were 3 for GAPDH and b-actin and 2 for spike in probe sets bioB, bioC and bioD on each array. The 3’-end to 5’-end probe intensity ratios were 0.93, 0.91, 0.92 and 0.94 for GAPDH and 4.47, 3.54, 3.89 and 3.82 for b-actin. Differences in percent of probe sets present between compared chips (determined by Affymetrix’s algorhythm) were below 10 percent.
