Comment[ArrayExpressAccession]	E-GEOD-54279
MAGE-TAB Version	1.1					
Public Release Date	2014-03-02
Investigation Title	Expression microarray analysis of human pancreatic islets reveals CD59 function					
Comment[Submitted Name]	Expression microarray analysis of human pancreatic islets reveals CD59 function
Experiment Description	Pancreatic islets are central in type 2-diabetes development, which coincides with increased activity of innate immunity. Intriguingly, human pancreatic islets express many complement genes. The most highly expressed gene was the complement inhibitor CD59 that is GPI anchored to the cell membrane, which unexpectedly was found in high amounts intracellularly in beta cells. Silencing of CD59 strongly suppressed insulin secretion. Importantly, this suppression was unrelated to established CD59 functions, but rather depletion of intracellular CD59. Imaging experiments identified a distal site of inhibition in the exocytotic pathway, but prior to emptying of the insulin granules. Proximity Ligation Assays pin-pointed the mechanism to impaired turnover of exocytosis-regulating SNARE-proteins and CD59 was detected in complex with VAMP2 and syntaxin. CD59 was downregulated by 24-h glucose incubations in human islets, rat cell lines and in islets from three rodent diabetes models. Islets from cadaver donors were provided by the Nordic Islet Transplantation Programme (www.nordicislets.org), Uppsala University. The microarrays were performed using GeneChipM-BM-. Human Gene 1.0 ST whole transcript according to Affymetrix standard protocol.					
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl				
Person Last Name	Storm	Storm	Groop			
Person First Name	Petter	Petter	Leif			
Person Email	petter.storm@med.lu.se					
Person Affiliation	Lund University					
Person Phone	+46-40 39 12 27					
Person Address	Lund University Diabetes Centre, Lund University, Jan WaldenstrM-CM-6ms gata 35, building 60, level 13, MalmM-CM-6, Sweden					
Person Roles	submitter					
Protocol Name	P-GSE54279-1	P-GSE54279-4	P-GSE54279-5	P-GSE54279-2	P-GSE54279-3	P-GSE54279-6
Protocol Description	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. ID_REF =  VALUE = log2 RMA normalized expression intensities	Briefly, 100-200 ng total RNA was processed as indicated by GeneChipM-. Expression 3M-bM-^@M-^Y-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 manufacturerM-bM-^@M-^Ys specifications.	Following fragmentation, 10 ug of cRNA were hybridized onto the GeneChipM-. Human Gene 1.0 ST whole transcript based assay overnight in the GeneChipM-. Hybridization oven 6400 using standard procedures. The arrays were washed and stained in a GeneChipM-. Fluidics Station 450.	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 M-5g/ml gentamicin, 0.25 M-5g/ml Fungizone (GIBCO, BRL, Gaithersburg, MD, USA), 20 M-5g/ml ciprofloxacin (Bayer Healthcare, Leverkusen, Germany), and 10 mM/l nicotinamide at 37 M-0C (5% CO2) for 1M-bM-^@M-^S9 days prior to RNA preparation.	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).	Scanning was carried out with the GeneChipM-. Scanner 3000 and image analysis was performed using GeneChipM-. Operating Software.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	HBA1C					
Experimental Factor Type	hba1c					
Comment[SecondaryAccession]	GSE54279					
Comment[GEOReleaseDate]	2014-03-02					
Comment[ArrayExpressSubmissionDate]	2014-01-22					
Comment[GEOLastUpdateDate]	2014-03-02					
Comment[AEExperimentType]	transcription profiling by array					
SDRF File	E-GEOD-54279.sdrf.txt