age: 66 bmi: 27.8 gender: male year of birth: 1946 tissue: pancreatic islets hba1c: 7.2
Treatment protocol
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 oligo package from bioconductor. Batch correction using COMBAT function from SVA package from bioconductor. Annotation using annotate package from bioconductor and hugene10sttranscriptcluster.db annotation data. The probesets were filtered as following (resulting a subest of 18883 ID_REFs out of a possible 33297): probsets that match uniquely to a gene in the latest hg19 human genome assembly, only probesets (or genes) that are on the autosomes, one probeset at random (if more than one probeset matches a gene) in order to have only 1 probeset to gene.
