|
| Status |
Public on Sep 01, 2008 |
| Title |
3.0_2A_3d_3VO |
| Sample type |
RNA |
| |
|
| Source name |
PCA ipsilateral, 3 days 3VO
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: sprague dawley rat
weight: 300-350g
age: 7 weeks
|
| Biomaterial provider |
HARLAN Winkelmann (Borchen, Germany)
|
| Treatment protocol |
Anesthesia was induced and maintained with 2% to 4% isoflurane (in oxygen). Analgesia was achieved by buprenorphine s.c. 0.1mg/kg intraoperatively and 0.05 mg/kg s.c. Vascular occlusions were carried out in one session by first electrocoagulating both vertebral arteries, using a paravertebral access (Pulsinelli and Brierley, 1979; Pulsinelli et al., 1983), followed by left common carotid artery ligation. After the wounds were closed animals were returned in pairs to their cages with free access to food and water and allowed to move freely. 3 days postoperatively anesthesia was induced by isoflurane (see above). Thereafter the vascular system was perfused with latex for visualization under the max. vasodilation by Papaverin. The brain was transferred into RNA Later for surgically extraction of the posterior cerebral artery. Posterior cerebral arteries of 8 animals were pooled for RNA extraction.
|
| Growth protocol |
Animals were housed under diurnal lighting conditions and allowed food and water ad libitum. Animal housing, care and applications of experimental procedures complied with the permission of state authorities according to the German Law for Protection of Animals, and the National Institute of Health Guidelines for Care and Use of Laboratory Animals. Conditions were in accordance with the recommendations of the Society of Laboratory Animal Science (GV-SOLAS) and the Federation of European Laboratory Animal Science Association (FELASA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Posterior cerebral arteries of 8 animals post 3d-3VO were pooled in one sample in RNAlater for RNA extraction. Total RNA was isolated using the RNeasy Mini-Kit (Qiagen, Hilden, Germany)
|
| Label |
biotin labeled
|
| Label protocol |
The amplification and labeling of the RNA samples were carried out according to the manufacturer´s instructions (Affymetrix, Santa Clara, CA). Briefly, total RNA was quantified by UV-spectroscopy and its quality evaluated by analysis on a LabChip (BioAnalyzer, AGILENT Technologies, Santa Clara, CA). In the presence of an oligo(dT)24 primer containing a T7 RNA polymerase promoter (TIBMOL Biol, Berlin, Germany) one to three micrograms of total RNA were used for double-stranded cDNA synthesis (SuperScript transcriptase, Life Technologies, Inc., Carlsbad, CA). Labeled complementary RNA (cRNA) was prepared from double-stranded cDNA by in vitro transcription using the GeneChip RNA transcript labeling kit (Affymetrix, Santa Clara, CA). After cleanup (Qiagen, Hilden, Germany), the biotin-labeled cRNA was fragmented by alkaline treatment (40mM Tris-acetate (pH 8.2), 100mM potassium acetate, and 50 mM magnesium acetate) at 94°C for 35 minutes.
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| |
|
| Hybridization protocol |
Fifteen micrograms of each cRNA sample was hybridized for 16 hours at 45°C to a rat expression Affymetrix RAE 230A GeneChip array. Following the hybridization arrays were washed and stained with streptavidin-phycoerythrin using a fluidics station in accordance with the Affymetrix protocol.
|
| Scan protocol |
Affymetrix GeneChip System confocal scanner 2500
|
| Description |
3 days upon 3VO surgery analysis of RNA extracted from the posterior cerebral artery
|
| Data processing |
Affymetrix GeneChip Operating Software (GCOS)
|
| |
|
| Submission date |
Oct 28, 2006 |
| Last update date |
Jun 23, 2008 |
| Contact name |
Philipp A.M. Hillmeister |
| E-mail(s) |
philipp.hillmeister@charite.de
|
| Phone |
0049-(0)30-450525034
|
| Fax |
0049-(0)30-450525934
|
| Organization name |
Charité Mitte Berlin
|
| Department |
Center for Cardiovascular Research
|
| Lab |
AG Buschmann
|
| Street address |
Hessische Straße 3-4
|
| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL341 |
| Series (1) |
| GSE6189 |
Molecular Mechanisms of Early Response in Adaptive Cerebral Arteriogenesis |
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