Comment[ArrayExpressAccession]	E-GEOD-58720
MAGE-TAB Version	1.1						
Public Release Date	2014-06-23
Investigation Title	Analysis of the effect of vascular endothelial growth factor on gene expression profile after focal cerebral ischemia in mouse						
Comment[Submitted Name]	Analysis of the effect of vascular endothelial growth factor on gene expression profile after focal cerebral ischemia in mouse
Experiment Description	To investigate the effect of vascular endothelial growth factor (VEGF) on gene expression profile after focal cerebral ischemia in mouse, we employed Agilent SurePrint G3 Mouse Gene Expression 8M-CM-^W60K Microarray as a platform to identify which genes influenced by VEGF in mouse after focal cerebral ischemia. Mice were randomized to sham group, in which mice underwent sham surgery; MCAO group, in which transient (90 min) middle cerebral artery occlusion (MCAO) model was performed and mice received vehicle (PBS, 0.01M, pH 7.4) intracerebroventricularly in the right lateral ventricle 3hr after reperfusion; VEGF group (n = 36), in which rhVEGF-A165 (10M-NM-<g/ml, dissolved in 0.01M PBS) was injected into the right lateral ventricle 3hr after reperfusion. Mice were sacrificed at 24hr after reperfusion, brains removed and peri-infarct areas were used for microarray. Gene expression microarray was applied to investigate the differentially expressed genes among sham group, MCAO group and VEGF group. Expression of thirty-seven genes was confirmed in the same RNA samples by real-time PCR. Gene expression in sham group, MCAO group and VEGF group was measured at 24 hours after reperfusion. Three independent experiments were performed using different mice for each experiment.						
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl					
Person Last Name	Wu	Wu	Jiang				
Person First Name	Li	Li	Yongjun				
Person Email	lilywu_yeah@aliyun.com						
Person Affiliation	Jinling Hospital, Nanjing University School of Medicine						
Person Address	Jinling Hospital, Nanjing University School of Medicine, 305 East Zhongshan Road, Nanjing, Jiangsu, China						
Person Roles	submitter						
Protocol Name	P-GSE58720-1	P-GSE58720-5	P-GSE58720-6	P-GSE58720-2	P-GSE58720-3	P-GSE58720-4	P-GSE58720-7
Protocol Description	The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. ID_REF =  VALUE = Normalized signal intensity	Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Agilent's Low Input Quick Amplification according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.	5 ug of Cy3-labelled cRNA was fragmented at 60M-0C for exactly 30 minutes in a reaction volume of 250 M-NM-<l containing 10 M-NM-<l 25x Agilent fragmentation buffer and 50 M-NM-<l 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 M-NM-<l of 2x GEx Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Mouse Gene Expression 8M-CM-^W60K Microarray for 17 hours at 65M-0C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37M-0C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.	Mice in sham group underwent sham surgery; mice in MCAO group underwent transient (90min) middle cerebral artery occlusion (MCAO) surgery and received 1M-NM-<l vehicle (PBS, 0.01M, pH 7.4) intracerebroventricularly in the right lateral ventricle 3hr after reperfusion; mice in VEGF group underwent 90min MCAO and received 1M-NM-<l rhVEGF-A165 (10M-NM-<g/ml, dissolved in 0.01M PBS) in the right lateral ventricle 3hr after reperfusion. Twenty-four hours after reperfusion, mice were sacrificed and brains were collected for microarray.	Adult male C57BL/6 mice weighing 23 to 25g (7~8 W) were housed in the same environment with 12 h light/dark cycle and relative humidity of 65%. The room temperature was maintained at approximately 25M-0C by air conditioning throughout all experiments. All animals were fasted overnight before the surgery, but were allowed free access to water.	Total RNA was extracted from homogenizing brains using TRIzolM-. Reagent (Invitrogen, Life Technologies, Carlsbad, California, USA) according to the manufacturerM-bM-^@M-^Ys protocol. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).	Slides were scanned immediately after washing on the Agilent Microarray Scanner (Agilent p/n G2565BA) using one color scan setting for 8M-CM-^W60K array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	PROTOCOL	DEVELOPMENTAL STAGE	COMPOUND				
Experimental Factor Type	protocol	developmental stage	compound				
Comment[SecondaryAccession]	GSE58720						
Comment[GEOReleaseDate]	2014-06-23						
Comment[ArrayExpressSubmissionDate]	2014-06-22						
Comment[GEOLastUpdateDate]	2014-06-24						
Comment[AEExperimentType]	transcription profiling by array						
SDRF File	E-GEOD-58720.sdrf.txt