|
| Status |
Public on Feb 22, 2011 |
| Title |
Sham413 |
| Sample type |
RNA |
| |
|
| Source name |
adult rat beta cells
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague Dawley
developmental stage: adult (6 weeks old)
|
| Treatment protocol |
Pancreas specimens obtained from adult and neonatal rats were embedded in Tissue-Tek OCT medium, immediately frozen in chilled isopentane, and stored at –80°C, pending sectioning at 8 μm. Frozen pancreatic sections were fixed in 70% ethanol for 30 seconds, rinsed in diethylpyrocarbonate (DEPC)-treated water for 30 sec, and dehydrated in 70% ethanol for 30 sec, 100% ethanol (rinsing), 100% ethanol twice for 1 min, and xylene for 4 minutes. Sections were air-dried and laser capture microdissection was performed. The microdissected cells were incubated with 10 μl of guanidine thiocyanate and polyethylene glycol octylphenol ether-based buffer for 30 minutes at 42°C, and the samples stored at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each population of laser captured beta cells using PicoPure RNA Isolation Kit (Arcturus) and amplified by T7-based linear amplification using T7-oligo-dT-primers. RNA amplification was performed using RiboAmp HS RNA Amplification Kit (Arcturus) following the manufacture’s protocol.
|
| Label |
biotin
|
| Label protocol |
Amplified RNA was converted into double-stranded complementary DNA (cDNA) using the RiboAmp HS RNA Amplification Kit (Arcturus), and biotinylated complementary RNA (cRNA) was generated from cDNA by in vitro transcription reaction using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics). RNA products were purified using the MiraColTM Purification Columns (Arcturus).
|
| |
|
| Hybridization protocol |
Biotinylated cRNA was fragmented to nucleotide stretches of 30-200 nucleotides and hybridized for 16hr at 45°C to the Affymetrix Rat Genome U34A Array. Following hybridization, arrays were washed and stained on the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
|
| Description |
Gene expression data from LCM excised adult rat beta cells
|
| Data processing |
Data were analyzed using DNA-chip Analyzer software (dChip), using the invariant set normalization algorithm.
The CEL file for this sample is missing 'STDV' and 'NPIXELS' data columns, and is readable only in dChip software. The file was renamed with ".txt" file extension because it does not conform to .CEL file specifications.
|
| |
|
| Submission date |
Oct 19, 2010 |
| Last update date |
Feb 22, 2011 |
| Contact name |
Susan Bonner-Weir PhD |
| E-mail(s) |
susan.bonner-weir@joslin.harvard.edu
|
| Organization name |
Joslin Diabetes Center
|
| Department |
Section on Islet Cell and Regenerative Biology
|
| Street address |
One Joslin Place
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL85 |
| Series (1) |
| GSE24790 |
Neonatal beta cells lack the specialized metabolic phenotype of mature beta cells |
|
