Gene Expression Analysis in an Animal Model of Cortical Dysplasia-CONTROL GROUP
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
SUBMITTER: Girish Hiremath
PROVIDER: E-GEOD-13676 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Cortical Dysplasia (CD) is the histopathological substrate in almost half of all drug-resistant epilepsy. Little is known about the gene expression profile of CD. As such information may help target therapeutics more effectively, our aim was to perform a gene expression analysis of an animal model of cortical dysplasia induced by in utero irradiation. THIS SERIES (GSE13697) INCLUDES ALL (AND ONLY) EXPERIMENTAL SAMPLES--I.E. IRRADIATED/CORTICAL DYSPLASIA (9). Nine offspring from irradiated animals, and nine age-matched controls were sacrificed at post-natal day 60. Cortex and hippocampal regions were separated, and total ribonucleic acid (RNA) was extracted using a commercially available kit (Qiagen®). RNA was then subjected to a gene expression analysis using an oligonucleotide microarray platform (Illumina®). After statistical analysis, genes were considered differentially expressed when a p value less than .001 was observed. Real-time, quantitative polymerase chain reaction (RT-qPCR) was used to confirm microarray results for three genes via the Livak method.
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Project description:We investigated the effects of PACAP treatment, and endogenous PACAP deficiency, on infarct volume, neurological function, and the cerebrocortical transcriptional response in a mouse model of stroke, middle cerebral artery occlusion (MCAO). PACAP-38 administered i.v. or i.c.v. one hour after MCAO significantly reduced infarct volume, and ameliorated functional motor deficits measured 24 hours later in wild-type mice. Infarct volumes and neurological deficits (walking faults) were both greater in PACAP-deficient than in wild-type mice, but treatment with PACAP reduced lesion volume and neurological deficits in PACAP-deficient mice to the same level of improvement as in wild-type mice. A 35,546-clone mouse cDNA microarray was used to investigate cortical transcriptional changes associated with cerebral ischemia in wild-type and PACAP-deficient mice, and with PACAP treatment after MCAO in wild-type mice. 229 known (named) transcripts were increased (228) or decreased (1) in abundance at least 50% following cerebral ischemia in wild-type mice. 49 transcripts were significantly up-regulated only at one hour post-MCAO (acute response transcripts), 142 were up-regulated only at 24 hours post-MCAO (delayed response transcripts) and 37 transcripts were up-regulated at both times (sustained response transcripts). More than 50% of these are transcripts not previously reported to be associated with brain ischemia responses. Many of the acutely regulated neurotrauma-associated transcripts, but a larger percentage of the delayed ones, require endogenous PACAP for up-regulation by ischemia, suggesting a more prominent role for PACAP in later response to injury than in the initial response, and consistent with a neuroprotective role for PACAP in late response to injury and neuroprotective efficacy when given post-MCAO. Putative injury effector transcripts regulated by PACAP include ãÃ-actin, midline 2, and metallothionein 1. Potential neuroprotective transcripts include several demonstrated to be PACAP-regulated in other contexts. Prominent among these were transcripts encoding the PACAP-regulated gene Ier3, and the neuropeptides enkephalin, substance P (tachykinin 1), and neurotensin. In the hybridization method employed, equal amounts (2-5ãÃg/15.5 ãÃl) of total RNA obtained from brain tissue of different experimental groups were separately labeled with two different fluorescent dyes (Cy3 or Cy5) and applied on the same NIMH Mouse 36K cDNA microarray chip containing 13217 distinct Entrez gene IDs (formerly known as LocusLink) and 15888 distinct unigene IDs, the remainders are incompletely annotated IMAGE clones. Probe preparation and hybridization were performed as follows. The total RNA (15-50 ãÃg in 15.5 ãÃl) was incubated with amine-modified random primer (2 ãÃg/ãÃl, 2 ãÃl) and RNase inhibitor (5 units/ãÃl, 1 ãÃl) at 70 äaC for 10 min. Primer-RNA solution was then incubated at 42 äaC for 2 hr with the reverse transcriptase mix containing 5X first-strand buffer, 50X aa-dUTP/dNTPs (25 mM dATP, 25 mM dGTP, 25 mM dCTP, 15 mM dTTP and 10 mM aminoallyl dUTP), 0.1 M DDT, and Superscript II reverse transcriptase. The cDNA were labeled with NHS-ester Cy3 or Cy5 dye in the presence of 1 M sodium bicarbonate. Array slides placed in a hybridization chamber (Corning, Corning, NY) were incubated at 42 äaC for 16-24 hr, and successively washed with 0.5X SSC, 0.01% SDS, and 0.06X SSC at room temperature for 10 min each. Arrays were scanned with a GenePix 4000A scanner (Axon, Foster City, CA), and the resulting images were analyzed using IPlab (Fairfax, VA) and a FileMaker Pro 5 (Santa Clara, CA) relational database designed by Zedong Chen, NHGRI. Following background subtraction and normalization, a calculated ratio of Cy3 to Cy5 signal intensities was used to define the relative increase or decrease of a particular transcript. Ratios were calculated only from those spots with a combined ratio quality above 0.3.
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