Genechip rat genome 230 2.0 array

Hippocampi were isolated from brains of MK-801-treated animals (n=12) and controls (n=12), shock-frozen in liquid nitrogen and immediately stored at –80 °C until further processing. Tissue (30–80 mg) was homogenized in QIAzol Lysis Reagent (Qiagen, Hilden, Germany) using ultra thurrax. Total RNA was extracted with the RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's instructions and stored at –80 °C. RNA concentration and quality was estimated using a UV spectrometer and an ethidium bromide-stained agarose gel. Equal amounts of RNA of three animals conferring to one experimental group were pooled, and 4.07±0.7 μg RNA of each of the resulting eight pools (four pools of three MK-801-treated animals and four pools of three control animals) hybridized to one array (GeneChip Rat Genome 230 2.0 Array, Affymetrix, Santa Clara, CA, USA). Expression data were normalized using the robust multiarray average method in GenePattern,³² and P-values were established using a two-tailed t-test.

Whole genome gene expression analysis was performed with the GeneChip Rat Genome 230 2.0 array (Affymetrix, Santa Clara, CA), which contains more than 28,000 rat genes, according to the instructions of the manufacturer. In brief, 150 ng of total RNA from each independent sample pool were transcribed to cDNA, amplified, and subjected to hybridization using the Affymetrix Fluidic Station 450 (Affymetrix, Santa Clara, CA) and the hybridization oven under standard conditions.
The probe-level signal data obtained from scanned chip images (GeneChip 7G Scanner; Affymetrix) were summarized and normalized using the Robust Multi-array Average (RMA) algorithm.

Total RNA was isolated from each immersed hepatic piece, WAT, and BAT by TRIzol reagent (Invitrogen Japan, Tokyo, Japan) and purified using RNeasy mini kits (Qiagen). Anti-sense RNA was synthesized from 100 or 200 ng of purified total RNA, and biotinylated complementary RNA (cRNA) was obtained using a GeneChip 3’IVT Express Kit (Affymetrix, Santa Clara, CA, USA). The cRNA was fragmented and hybridized to a GeneChip Rat Genome 230 2.0 Array (Affymetrix) for 16 h at 45 °C. The arrays were washed and stained with phycoerythrin using the GeneChip Fluidics Station 450 (Affymetrix) and submitted to scanning on an Affymetrix GeneChip Scanner 3000 7G. The Affymetrix GeneChip Command Console Software was used to make CEL files.

Microarray profiling was outsourced to Expression Analysis (Durham, NC). Biotinylated cRNA was generated from 100 ng of total RNA using the GeneChip 3′ IVT Express Target Labeling method (Affymetrix, Santa Clara, CA). For each sample, 10 ug of biotinylated cRNA spiked with hybridization controls (bioB, bioC, bioD and cre) was hybridized to an Affymetrix GeneChip® Rat Genome 230 2.0 array for 16–18 hours at 45°C. Following hybridization, arrays were washed and stained in a GeneChip® Fluidics Station and scanned with a GeneChip® Scanner 3000 (Affymetrix). Quality checks and data analyses were carried out using Affymetrix GeneChip Operating Software (GCOS).

Total RNA was extracted from the cells using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Any contaminating deoxyribonucleic acids were removed via DNase I digestion. The Affymetrix GeneChip Rat Genome 230 2.0 Array was used for the microarray analysis. Three independent cell samples were used in the microarray analysis (n = 3). Hybridization, data acquisition, and analyses were performed by the CapitalBio Corporation (Beijing, China). The microarray results were analysed using the robust multiarray average (RMA) method. Significance analysis of microarrays (SAM) was used to identify differentially expressed genes. SAM-identified genes with statistically significant expression changes using a set of gene-specific t-tests and a permutation process provided an estimated false discovery rate (FDR) from the generated data. Genes with scores higher than the threshold value or genes with FDR values lower than the threshold value were considered significant. Additionally, fold-change analysis was performed to calculate the ratios of the geometric means of expression intensities. To select differentially expressed genes, we used ≥1.5- and ≤1.5-fold changes as threshold values and a significance level of <5% FDR. Signalling pathway analyses were performed for the genes with significant differences using the DAVID online analysis tool.

The cortex or peri-infarct cortex was harvested and rinsed with cold PBS containing diethyl pyrocarbonate for eliminating RNase. Analysis was performed using an Affymetrix GeneChip^® Rat Genome 230 2.0 Array (31,000 probe set). RNA sample preparations and microarray hybridization procedures were performed by CapitalBio Corporation (Beijing, China), according to the procedures outlined by Affymetrix.

RNA was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, U.S.A). Double-stranded cDNA was synthesized from total RNA. An in vitro transcription was performed to produce biotin-labeled cRNA from cDNA. Finally, cRNA was hybridized to a GeneChip Rat Genome 230 2.0 Array (Affymetrix, CA, U.S.A) for 16 hours at 45°C. This microarray chip provides the entire transcribed rat genome on a single array, and analyzes more than 31,000 genes at one time. After hybridization, chips were washed and dried, and then scanned by the GeneChip Scanner 3000 (Affymetrix). To calculate the fold change, the expression value of each gene from the compression group was divided by the expression value from the sham group. The whole microarray working process was accomplished at the Analysis Center of Life Science, Hiroshima University. Biological information of each probe was obtained from NetAffx Analysis Center (http://www.affymetrix.com/analysis/index.affx).