Affymetrix GeneChip Murine Genome U74Av2 [MG_U74Av2]

Discovery of transcriptional programs in cerebral ischemia by in silico promoter analysis

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profiling gene transcription in a mouse model of permanent focal cerebral ischemia that was induced by middle cerebral artery occlusion (MCAO)

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Transcription profiling of mouse forebrain with induced middle cerebral artery occlusion (MCAO) to study permanent focal cerebral ischemia

profiling gene transcription in a mouse model of permanent focal cerebral ischemia that was induced by middle cerebral artery occlusion (MCAO)


Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-TABM-3535

For microarray experiments mice were re-anaesthetized at the times indicated and perfused with Ringer solution. Brains were quickly removed and frozen on dry ice. After removal of cerebellum, brain stem, and olfactory bulb, the forebrains were halved. RNA was prepared by a guanidinium-isothiocyanate acid-phenol extraction, after homogenizing the tissue with an Ultraturrax T25 (Ilka Labortechnik, Staufen, Germany). This step was followed by further purification using RNAeasy columns (Qiagen, Hilden, Germany). RNA concentrations were determined by photometric measurements (DU640, Beckman Coulter, Fullerton, USA) and samples were stored at ﾖ80ï¾°C until used. We checked RNA quality with the Agilent 2100 Bioanalyzer.

P-TABM-3536

First and second strand cDNAs were synthesized from 10 ?g of total RNA using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) and oligo-dT24-T7 (5'-GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG-3') primer according to the manufacturer's instructions. cRNA was synthesized and labeled with biotinylated UTP and CTP by in vitro transcription using the T7 promoter coupled double stranded cDNA as template and the T7 RNA Transcript Labeling Kit (ENZO Diagnostics Inc.).

P-TABM-3537

Male C57Bl/6 mice at an age of 3 to 4 months were used.

P-TABM-3538

Affymetrix standard normalization by MAS5

P-TABM-3539

C57Bl/6 mice were anaesthetized with  halothane (1%) and N2O/O2 (70%/30%) and an 8-0 nylon suture (Dermalon, Braun-Dexon, Spangenberg, Germany) coated with Xanthopren M Mucosa (Heraeus Kulzer, Dromagen, Germany) was inserted into the common carotid artery on the left side and advanced into the internal carotid artery until the laser Doppler signal decreased considerably to occlude the middle cerebral artery (MCAO). After 90 min of occlusion the filament was retracted to allow for reperfusion. Mice were sacrificed after 2, 6 or 20 minutes of reperfusion. For sham surgery, preparation of the common carotid artery was identical but the suture was not inserted.

Transcription profiling of mouse forebrain with induced middle cerebral artery occlusion (MCAO) to study permanent focal cerebral ischemia

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