|
| Status |
Public on Dec 04, 2013 |
| Title |
dentate gyrus-granule cell layer-30daysRunning, repAT1 |
| Sample type |
RNA |
| |
|
| Source name |
Outer granule cell layer from a mouse allowed access to running wheel for 30 days
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 10-11 week old
Sex: male
tissue: brain
region: dentate gyrus-granule cell layer
days_running: 30
animal: AT1
|
| Treatment protocol |
Brains were quickly removed, flash-frozen in OCT mounting medium in a dry ice-isopentantane slurry, and stored at -80C until use. 12μm thin cryostat sections were stained with cresyl violet, rapidly dehydrated through xylenes, and stored until use a vacuum dessicator. An Arcturus PixCell II machine (Arcturus, Mountain View, CA) was used to isolate 2-3 cell thick bands from both the inner and outer portions of the dentate granule cell layer.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified from the laser captured tissue using the Absolutely RNA Nanoprep Kit (Stratagene, La Jolla, CA), and RNA yield was assessed using the RiboGreen RNA Quantitation Reagent (Molecular Probes Inc, Eugene, OR). RNA from three groups of animals (no running, 4 days of running, 30 days of running), consisting of 3 animals each, was isolated independently and amplified separately.
|
| Label |
biotin
|
| Label protocol |
5ng total RNA starting material was amplified using the MessageAmp aRNA Kit (Ambion Inc, Austin, TX). This method utilizes reverse transcription with T7 RNA Polymerase. The first and second rounds of the kit were performed per the manual, with an additional third round identical to the second round except that the in vitro transcription step utilized the Enzo BioArray High Yield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA).
|
| |
|
| Hybridization protocol |
15μg of biotinylated aRNA was fragmented and hybridized to Affymetrix MG-U74Av2 arrays.
|
| Scan protocol |
Gene chips were scanned following the manufactures protocol.
|
| Description |
Gene expression data from 10- to 11-week-old C57BL/6 male mice
|
| Data processing |
Initial data analysis and normalization were done using the Affymetrix Expression Console using two algorithms: Robust Multichip Analysis (RMA) and Probe Logarithmic Intensity Error Estimation (PLIER) using the minus mismatch option (-MM). Note that an additional filtering step to remove the effect of running was performed in the larger cross-species study, using ComBat (www.bu.edu/jlab/wp-assets/ComBat).
|
| |
|
| Submission date |
Jul 27, 2012 |
| Last update date |
Dec 04, 2013 |
| Contact name |
Jeremy Miller |
| E-mail(s) |
[email protected]
|
| Organization name |
Allen Institute for Brain Science
|
| Street address |
615 Westlake Ave N
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL81 |
| Series (1) |
| GSE39697 |
Molecular Signatures of Neurogenesis in the Hippocampal Subgranular Zone of Rodents |
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