strain background: CBA-C57BL/6 genotype/variation: pIns-MYCERTAM transgenic gender: male age: 8-12 weeks tissue: pancreatic islets treated with: peanut oil (vehicle for 4OHT) treated with: water (vehicle for exendin-4)
Treatment protocol
All mice were housed and treated in accordance with protocols and regulations sanctioned by the Home Office under the Animals Act of 1986. Mice used throughout studies were adults, aged 3 months at the start of studies. Activation of MYCERTAM was achieved through administration of 1mg of 4 hydroxytamoxifen (4OHT; Sigma-Aldrich, St. Louis, MO) by daily intraperitoneal injection. To assess the effect of exendin-4 on MYCER-induced hyperglycaemia, mice were given either twice-daily subcutaneous (sc) injections of exendin-4 (50ug/kg dissolved in 5mls water), or equivalent volumes of water vehicle, starting 2 days prior to 4OHT injections.
Growth protocol
Pelengaris S, Khan M, Evan G (2002) Suppression of Myc-induced apoptosis in beta cells exposes multiple innate oncogenic properties of Myc sufficient to trigger immediate carcinogenic progression. Cell 109: 321-334
Extracted molecule
total RNA
Extraction protocol
Fresh frozen pancreas sections were cut to a thickness of 15 µm, bound to a MMI MembraneSlide (Molecular Machines and Industries, Rockledge, FL) and fixed in ice-cold 100 % ethanol for 2 minutes. The SL µCut laser capture microdissection system (Molecular Machines and Industries, Rockledge, FL) was used to isolate islets from surrounding exocrine tissue. Isolated islets were collected on the lid of a MMI IsolationCap (Molecular Machines and Industries, Rockledge, FL) and RNA was homogenised in a solution of RLT buffer (Qiagen, Valencia, CA), a guanidine-isothiocyanate-containing lysis buffer, with β-mercaptoethanol as described in the RNA Microkit protocol. Total RNA was isolated from homogenised pancreata lysates using the Qiagen Microkit (Qiagen, Valencia, CA) for small sample RNA preparation, incorporating a DNase I treatment step. RNA integrity was analysed using an Agilent 2100 Bioanalyser (Agilent, Santa Clara, CA) and RNA was quantified using an Ambion Nanodrop spectrophotometer (Ambion, Foster City, CA).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
10µg double-amplified biotin-labelled cRNA were hybridised to Affymetrix MOE430 2.0 GeneChips (Affymetrix, Santa Clara, CA) together with pre-labelled hybridisation controls as described in the Affymetrix GeneChip Expression Analysis Technical Manual.
Scan protocol
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
Description
MycOFF_water_8_2
Data processing
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. Further processing was performed by GCRMA normalization. After the quality control step, the following 8 samples out of 60 showing poor reproducibility were excluded from our further study: GSM930242, GSM930247, GSM930251, GSM930263, GSM930264, GSM930289, GSM930291, GSM930298
