homo sapiens, CCRF-CEM (ATCC no.: CLL-119) subclone C7R1 (=GC-resistant) transfected with a dimerization deficient Glucocorticoid-receptor-mutant, high expression of mutant resulting in a restored GC-sensitivity, cultured under standard conditions (37°C, 5% CO2, saturated humidity), in the presence of ethanol 0,1% for 6h
Growth protocol
cultured under standard conditions (37°C, 5% CO2, saturated humidity)
Extracted molecule
total RNA
Extraction protocol
For total RNA isolation, TRIreagent (MRC Inc., Cincinnati, OH,USA) was used according to the manufacturer’s protocol. Briefly, up to 1x10E7 cells per ml were lysed with TRIreagent, 200ul of chloroform were added, the mixture centrifuged, and the RNA from the aqueous phase precipitated with isopropanol. The pelleted RNA was washed in 70% ethanol in DEPC-water and resuspended in nuclease-free water. RNA quantity and purity was determined by optical densitiy measurements (OD260/280) and RNA inte Glucocorticoid-receptority by using the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA,USA), and only high quality RNA was further processed.
Label
R-Phycoerythrin
Label protocol
The chips were stained and washed in an Affymetrix fluidic station 450 following the EukGE-ws2v4 protocol.
Hybridization protocol
On rotation (60 rpm) hybridization at 45C for 16 hours
Scan protocol
Fluorescence signals were recorded by an Affymetrix scanner 3000 and image analysis performed with the GCOS software (version 1.2).
Description
biological source: homo sapiens, CCRF-CEM (ATCC no.: CLL-119) subclone C7R1 (=GC-resistant) transfected with a dimerization deficient Glucocorticoid-receptor-mutant, high expression of mutant resulting in a restored GC-sensitivity, cultured under standard conditions (37°C, 5% CO2, saturated humidity), in the presence of ethanol 0,1% for 6h
Data processing
Data processing and analysis was performed in R using Bioconductor version 1.5. GeneChip raw expression values were normalized and summarized using the robust multiarray analysis (RMA) method proposed by R. Irizarry.
