Investigation Title Transcription profiling of mouse cerebral cortex and striatum after transient focal ischemia in ovarectomized animals to investigate estrogen response after stroke Comment[Submitted Name] Estrogen response after stroke Experimental Design compound_treatment_design transcription profiling by array Experimental Design Term Source REF mo EFO Comment[ArrayExpressReleaseDate] 2008-06-13 Comment[SecondaryAccession] GSE5315 Comment[SecondaryAccession] GDS2311 Comment[AEMIAMESCORE] 4 Comment[ArrayExpressAccession] E-GEOD-5315 Comment[MAGETAB TimeStamp_Version] 2010-08-06 15:49:23 Last Changed Rev: 13058 Experimental Factor Name Compound Experimental Factor Type compound Experimental Factor Term Source REF Person Last Name Alkayed Person First Name Nabil Person Mid Initials J. Person Email Person Phone Person Fax Person Address Anesthesiology & Peri-Operative Medicine, Oregon Health & Science University, 3181 S.W. Sam Jackson Pk. Rd., UHS-2, Portland, 97239-3098, USA Person Affiliation Oregon Health & Science University Person Roles submitter Person Roles Term Source REF Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2008-06-13 PubMed ID 16971488 Publication DOI 16971488 Publication Author List Yun Xu, Wenri Zhang, Judith Klaus, Jennifer Young, Ines Koerner, Laird C Sheldahl, Patricia D Hurn, Francisco Martínez-Murillo, Nabil J Alkayed Publication Title Role of cocaine- and amphetamine-regulated transcript in estradiol-mediated neuroprotection. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine- and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases. Experiment Overall Design: affy platform; replicate samples; two treated samples, two placebo. Experiment Overall Design: MIAME compliance: All samples were run in commercial arrays from Affymetrix, using Rat Genome U34A GeneChip arrays as described in the Affymetrix web site. The sample information is described below. Likewise, the Affymetrix protocol is detailed and referenced above. These descriptions include all information currently considered under the MIAME supportive guidelines, with which the JHMI Microarray Core Facility abides in all its procedures. Experiment Overall Design: Methodology. Expression profiling using DNA microarrays (One round amplification): Experiment Overall Design: Experimental Design and Sample Information. Transient focal cerebral ischemia was induced in ovariectomized female rats with and without replacement with 17beta-estradiol by intraluminal middle cerebral artery occlusion (MCAO), as described in the manuscript. At 6 and 24 hours after 2-hour MCAO, total RNA was prepared from the ischemic and contralateral cerebral cortex and striatum between coronal levels 2 and 3 mm relative to Bregma, which represent the core of MCA territory. RNA samples from 3 animals were combined into one pooled sample and hybridized to Affymetrix RG-U34A GeneChip, as described below. Each microarray experiment was performed in duplicate using two independent pooled samples per group (6 animals per group at each time point, 2 pooled samples for each brain region per time point). Experiment Overall Design: Microarray Hybridization Protocol. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN). RNA from control and experimental tissue are processed using the RNA amplification protocol described by Affymetrix (Affymetrix Expression Manual). Briefly, 5 micrograms of total RNA were used to synthesize first strand cDNA using oligonucleotide probes with 24 oligo-dT plus T7 promoter as primer (Proligo LLC, Boulder, Colorado) and the SuperScript Choice System (Invitrogen, Carlsbad, California). Following the double stranded cDNA synthesis, the product was purified by phenol-chloroform extraction, and biotinilated anti-sense cRNA was generated through in vitro transcription using the BioArray RNA High Yield Transcript Labeling kit (ENZO Life Sciences Inc., Farmingdale, New York). 15 ug of the biotinilated cRNA were fragmented at 94 C for 35 min (100mM Tris-acetate, pH 8.2, 500mM KOAc, 150mM MgOAC), and 10ug of total fragmented cRNA were hybridized to the Affymetrix U34A GeneChip arrays for 16hr at 45 C with constant rotation (60 rpm). Affymetrix Fluidics Station 450 was then used to wash and stain the Chips, removing the non-hybridized target and incubating with a streptavidin-phycoerythrin conjugate to stain the biotinilated cRNA. The staining was then amplified using goat IgG as blocking reagent and biotinilated anti-streptavidin antibody (goat), followed by a second staining step with a streptavidin-phycoerythrin conjugate. Experiment Overall Design: Quality Control. To ascertain the quality control of the total RNA from the samples, we used the Agilent Bioanalyzer, Lab on a Chip technology, and confirmed the rRNA ratios and clean run patterns of the samples. Likewise, this technology is used to confirm the quality of the RNA in the form of cRNA and fragmented cRNA. To assess the QC of the hybridization, GeneChip image, and comparison between chips, we confirmed the following parameters: scaling factor values within comparable range; low background values (50-95); high percentage of present calls (between 30 and 55%); consistent 3’/5’ ratios of GAPDH as representation of housekeeping genes, and presence or absence of Bio B and C as internal spike controls. Protocol Name Protocol Type Protocol Description Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF SDRF File E-GEOD-5315.sdrf.txt Term Source Name ncbitax The MGED Ontology ArrayExpress NCI_thesaurus mo EFO The MGED Ontology Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress ncithesaurus.obo.alt http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version