Comment[ArrayExpressAccession] E-MTAB-3117 MAGE-TAB Version 1.1 Investigation Title Exendin-4 treatment of Pancreatic Islets Comment[Submitted Name] Exendin-4 treatment of Pancreatic Islets Experiment Description The aim of the study was to investigate the effect of Exendin-4 on isolated pancreatic islets allowing for the elucidation of the various transcriptional programs initiated by this multifunctional peptide hormone. Islets were treated with Exendin-4 for 30 and 180 minutes. After these time points, RNA was isolated and used for hybridization against matched control islets using the Mouse PancChip 6. Experimental Design compound treatment design time series design Comment[AEExperimentType] transcription profiling by array Experimental Factor Name compound time Experimental Factor Type compound time Public Release Date 2014-11-13 Person Last Name Habener Person First Name Joel Person Mid Initials Person Email jhabener@partners.org Person Phone Person Address 55 Fruit Street, Boston, MA, 02114, USA Person Affiliation Massachusetts General Hospital Person Roles submitter PubMed ID Publication Author List Publication Title Publication Status Protocol Name P-MTAB-42210 P-MTAB-42211 P-MTAB-42212 P-MTAB-42213 P-MTAB-42214 P-MTAB-42215 P-MTAB-42216 P-MTAB-42217 P-MTAB-42218 Protocol Type purify incubate compound based treatment nucleic acid extraction protocol linear_amplification nucleic acid labeling protocol hybridization image_acquisition feature_extraction Protocol Description Islets of Langerhans were isolated from fed 7-8 week old C57Bl/6 mice (Jackson Laboratories, Bar Harbor ME) by collagenase digestion (collagenase type VI, Sigma) at 37C using a physiological saline solution supplemented with 1mM CaCl2 and 4mM glucose and equilibrated with 5% CO2/ 95% O2, pH 7.4. Islets were handpicked using a binocular microscope. Groups of 50 islets were incubated in physiological saline solution supplemented 11mM glucose, and 1% BSA. After incubation of either (30, 60 or 180 min), the islets were centrifuged for 5 minutes at 5,000rpm. Groups of 50 islets were incubated in physiological saline solution supplemented 11mM glucose, 1% BSA, and 10nM Exendin-4. After incubation of either (30, 60 or 180 min), the islets were centrifuged for 5 minutes at 5,000rpm. Adherent cells were rinsed with Phosphate Buffered Saline (PBS), and cells were scraped off the plates and lyzed using the buffer RLT of the RNA isolation Kit from Qiagen (RNeasy). They were then placed in a freezer for a specified amount of time at a specified temperature.(Total) RNA was isolated following manufacturer's protocol including an optional DNAse treatment using the RNAse free DNAse kit from Qiagen. RNA is amplified using the MessageAmp aRNA Kit (Ambion, Austin, TX). Total RNA is reverse transcribed using a T7 Oligo(dT) Primer and reverse transcriptase at 42C for 2 hours. Second-strand synthesis is performed using DNA Polymerase. The resulting cDNA is purified with a filter cartridge. Double-stranded cDNA is concentrated and in vitro transcribed at 37 C to make antisense RNA (aRNA). The aRNA is DNase-treated, purified with a filter cartridge, and eluted with nuclease-free water. 2 microg of amplified mRNA was reverse transcribed to incorporate aminoallyl dUTP (sigma) into the cDNA for each sample. The reaction was purified (QIAquick PCR Purification kit, Qiagen) and then coupled to the monofunctional NHS ester Cy3 or Cy5 (Amersham Biosciences) for 1hour and then both reactions were combined. The combined labeled cDNA was hybridized onto the mouse PancChip v6.0 microarray (A-CBIL-9) and incubated using a hybridization chamber (Genisphere) AT 42C for 16 hours. The arrays were then washed briefly. Settings: laser power=100%. Settings: software version=4.0.1.23; background density measure=local; feature layout=rectangular; standard deviation=normal; feature diameter=120 um;number of block rows=12;number of block columns=4;number of feature rows=17; number of feature columns=18. Protocol Term Source REF MGED MGED EFO EFO MGED EFO MGED MGED MGED Protocol Hardware GenePix 4000B Protocol Parameters incubation time incubation time pmt Term Source Name EFO ArrayExpress MGED Term Source File http://www.ebi.ac.uk/efo http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php Comment[AdditionalFile:tif] EX30MIN1_Cy3Cy5.tif Comment[AdditionalFile:tif] EX30MIN2_Cy3Cy5.tif Comment[AdditionalFile:tif] EX30MIN3_Cy3Cy5.tif Comment[AdditionalFile:tif] EX30MIN4_Cy3Cy5.tif Comment[AdditionalFile:tif] EX30MIN5_Cy3Cy5.tif Comment[AdditionalFile:tif] EX180MIN1_Cy3Cy5.tif Comment[AdditionalFile:tif] EX180MIN2_Cy3Cy5.tif Comment[AdditionalFile:tif] EX180MIN3_Cy3Cy5.tif Comment[AdditionalFile:tif] EX180MIN4_Cy3Cy5.tif Comment[AdditionalFile:tif] EX180MIN5_Cy3Cy5.tif SDRF File E-MTAB-3117.sdrf.txt