cell type: human islet beta cells treatment: IFN-beta and IFN-gamma time: 48h
Treatment protocol
Following the initial culture period, islets were cultured for the indicated time points (1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120,132, 144 and 168 hours.) at 5.6 mM glucose RPMI-1640 containing 10% FCS and recombinant human IL-1β (25 units/ml) (PeproTech, London, U.K.) and IFN-γ (1,000 units/ml) (PeproTech) before being retrieved for RNA extraction and array analysis. The culture media were changed on days 2 and 5.
Growth protocol
The human pancreatic islets were isolated from the pancreas of brain dead organ donors using collagenase digestion and Biocoll gradient centrifugation. After isolation the islets were pre-cultured free floating in Sterilin dishes in RPMI 1640 medium containing 5.6 mM glucose, 10% FCS and 2 mM Lglutamine for 3-5 days.
Extracted molecule
total RNA
Extraction protocol
Following the different periods of cytokine exposure, RNA was prepared from 500-1000 islets/group using the Phenol-Chaosolv method (ULTRASPEC RNA isolation system from Biotecx).
Label
biotin
Label protocol
standard Affymetrix protocol
Hybridization protocol
standard Affymetrix protocol
Scan protocol
The output of microarray experiments (Affymetrix Human Genome U133 Plus 2.0 array) were stored in DAT files, processed by Affymetrix software to perform background subtraction and saved as .CEL files, containing intensity values for the probes in the chip.
Data processing
The pre-processing of the .CEL files was done using Custom Chip Definition (CDF) files, based on ENTREZ gene Ids (platform hgu133plus2). The R package simpleaffy was used to read the data.
