|
| Status |
Public on Oct 05, 2011 |
| Title |
blood-saline+ischemic challenge-24h-3 |
| Sample type |
RNA |
| |
|
| Source name |
blood, saline+ischemic challenge, 24h
|
| Organism |
Mus musculus |
| Characteristics |
tissue: whole blood
strain: C57/Bl6
treatment: saline+ischemic challenge 24h
|
| Treatment protocol |
Groups of C57Bl6 mice (n=4/treatment/time for all except sham+ischemic challenge (n=3/treatment/time)) received either preconditioning alone, preconditioning plus ischemic challenge (45 min MCAO) or ischemic challenge alone. Preconditioning paradigms included: LPS (0.2 mg/kg; ip), CpG (0.8 mg/kg; ip), saline (ip), brief ischemia (MCAO, 12 min) or sham surgery (duplicates time and procedure for brief ischemia with out suture advancement). For groups receiving preconditioning alone, mice were euthanized for blood and tissue collection at 3, 24 and 72 hr post preconditioning. In groups receiving preconditioning plus ischemic challenge, MCAO was performed 72 hr following the preconditioning stimulus and mice were euthanized for blood and tissue collection at either 3 or 24 hr post occlusion. Groups receiving ischemic challenge alone received 45 min MCAO with no prior pretreatment and tissue was collected at 3 and 24 hr post occlusion. Blood and tissue were also collected from 6 unhandled mice to include as a baseline control group. At the time of euthanization all mice were anesthetized, then perfused with heparinized saline. Under RNase-free conditions the ipsilateral cortex region from the frontal 4 mm of brain was isolated and snap frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For brain samples: Total RNA was isolated using the Qiagen RNeasy Lipid tissue mini kit (Qiagen Inc.).
For blood samples: Total RNA was isolated from fresh blood using the QIAamp RNA Blood Mini Kit (Qiagen Inc.).
|
| Label |
biotin
|
| Label protocol |
For brain samples: Fifty ng of each brain RNA sample were labeled with Ovation RNA Amplification System V2 and FL-Ovation cDNA Biotin Module V2 (NuGEN Technologies, San Carlos, CA).
For blood samples: Fifty ng of each blood RNA sample were prepared for hybridization using a modification of the standard Ovation protocol that has been optimized for working with whole blood RNA. This protocol, described as the Ovation Whole Blood Solution (NuGEN Technologies), uses the Ovation RNA Amplification System V2 with Ovation WB Reagent for the cDNA synthesis, RNA amplification steps of the target labeling protocol.
|
| |
|
| Hybridization protocol |
Labeled target was fragmented at 95o C in the presence of high magnesium concentration. Ten μg of target was hybridized with the GeneChip MOE430 2.0 array (Affymetrix) overnight, followed by washing, staining with streptavidin-phycoerythrin (Molecular Probes), signal amplification with biotinylated anti-streptavidin antibody (Vector Labs), and a final staining step on the Fluidics Station 400 (Affymetrix).
|
| Scan protocol |
The distribution of fluorescent material on the processed array is determined using the Agilent GeneArray laser scanner (Affymetrix). Image inspection is performed manually immediately following each scan. Software- Affymetrix GCOS v. 1.4.0.036. Scanning hardware- Agilent Gene Array Scanner
|
| Description |
corresponds to brain sample ICC5
|
| Data processing |
Probe level measurements produced by GCOS (.cel files). The brain and blood data were normalized seperately using the Robust Multichip Average method.
|
| |
|
| Submission date |
Sep 30, 2011 |
| Last update date |
Oct 05, 2011 |
| Contact name |
Susan L Stevens |
| E-mail(s) |
stevensu@ohsu.edu
|
| Organization name |
Oregon Health & Science University
|
| Department |
Molecular Microbiology & Immunology
|
| Lab |
Mary Stenzel-Poore
|
| Street address |
3181 SW Sam Jackson Pk Rd
|
| City |
Portland |
| State/province |
OR |
| ZIP/Postal code |
97239 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE32529 |
Mouse ischemic tolerance genomic analysis of the brain and blood. |
|
