strain: male wistar rats weight: 160-200g age: 7 weeks
Biomaterial provider
HARLAN Winkelmann (Borchen, Germany)
Treatment protocol
stroke operated: Five days after beginning of treatment, focal cerebral ischemia was induced by right middle cerebral artery occlusion (MCAO) for 90min with subsequent reperfusion as described previously (Groth et al., 2003; Dai et al., 1999). pre-treatment: vehicle (0.9% NaCl) twice daily s.c. for five days
Growth protocol
Animals were kept in a SPF barrier under standardisized conditions with respect to temperature and humidity, and were housed on a 12h light/12h dark cycle in groups of 4-5 with free access to food and water. Animal housing, care, and applications of experimental procedures complied with the Guide for the Care and Use of Laboratory Animals of the Government of Berlin, Germany and are in accordance to the recommendations of the Society for Laboratory Animal Science (GV-SOLAS) and the Federation of European Laboratory Animal Science Associations (FELASA).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated 2 h after MCAO from a segment of the right hemisphere of the brain using TRIzol Reagent (Invitrogen).
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 (40 mM Tris-acetate (pH 8.2), 100 mM potassium acetate, and 50 mM magnesium acetate) at 94°C for 35 minutes.
Hybridization protocol
Fifteen micrograms of each cRNA sample was hybridized for 16 hours at 45°C to rat expression Affymetrix RAE 230A GeneChip arrays. 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
stroke operated animals with vehicle pre-treatment
