The islets were cultured in CMRL 1066 (ICN Biomedicals, Costa Mesa, CA, USA) supplemented with 10 mM/l HEPES, 2 mM/l L-glutamine, 50 µg/ml gentamicin, 0.25 µg/ml Fungizone (GIBCO, BRL, Gaithersburg, MD, USA), 20 µg/ml ciprofloxacin (Bayer Healthcare, Leverkusen, Germany), and 10 mM/l nicotinamide at 37 °C (5% CO2) for 1–9 days prior to RNA preparation.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany). RNA quality and concentration were measured using an Agilent 2100 bioanalyzer (Bio-Rad, Hercules, CA, USA) and a Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA).
Label
biotin
Label protocol
Briefly, 100-200 ng total RNA was processed as indicated by GeneChip® Expression 3’-Amplification Reagents Onecycle cDNA synthesis kit instructions (Affymetrix Inc, Santa Clara, CA, USA) was used to produce double-stranded cDNA. This was used as a template to generate biotintargeted cRNA following the manufacturer’s specifications.
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized onto the GeneChip® Human Gene 1.0 ST whole transcript based assay overnight in the GeneChip® Hybridization oven 6400 using standard procedures. The arrays were washed and stained in a GeneChip® Fluidics Station 450.
Scan protocol
Scanning was carried out with the GeneChip® Scanner 3000 and image analysis was performed using GeneChip® Operating Software.
Description
Gene expression data from human pancreatic islets
Data processing
The array data was summarized and normalized with Robust Multi-array Analysis (RMA) method using the software “Expression Console” (Affymetrix).
