Methods Animals All studies were reviewed and approved by IACUC committee of the University of California at Davis. All mice had food and water available ad libitum on a 12 hour light dark cycle, and were acclimated to the animal room for at least one week before the experiment. Hypoxia and Tissue Samples C57BL 6 male mice, ages 8 9 weeks, were exposed to selleck products 8% O2 and 92% N2 for 3 hours in hypoxia chambers and then returned to room air in their home cages for 24 hours. Sham treated control mice were also placed in the same chambers but with room air for 3h, and then returned to their home cages for 24h. During the experiments mouse brains were removed and dissected in a cold room at the fol lowing time points immediately after 1 hr of hypoxia, immediately after 3 hr of hypoxia, and after 1, 3, 6, 12, and 24 hours of re oxygenation.
Total RNA was purified from the following brain regions cerebral cortex, hippocampus, striatum, thalamus, midbrain, cerebellum and pons medulla. Three mice were studied for each hypoxia time point and sham condition, for a total of 24 mice studied and 168 samples. RNA and microarray Brain samples were homogenized in 1 ml Inhibitors,Modulators,Libraries of TRIZOL Reagent. RNA purification Inhibitors,Modulators,Libraries was carried out according to the man ufacturers recommendations. The RNA pellet was cleaned using a RNAeasy mini kit. RNA purity and integrity were assessed using a Nanodrop spectrophotometer and an Agilent Bioanalyzer. Samples were processed on whole genome Gene Chip Mouse Expression 430 2. 0 arrays according to the Affymetrix technical manual. Samples had to have an A260 A280 absorbance ratio greater than 1.
9 and a 28S 18S rRNA ratio greater than 1. 5. The raw data are available through NCBI Gene Expression Omnibus with series accession num ber. Microarray Data Analysis Raw signals were transformed into. CEL files in GCOS software. Probe data were generated using the Robust Multi chip Average with GC content Background Correction in Genespring Inhibitors,Modulators,Libraries 7 software. This involves back ground correction, quantile normalization, and summarization of the probe set values into gene level expression measurements. The expression data for each brain region of hypoxia treated mice were then normal ized to the averaged values for each brain region from the sham treated mice. Differentially regulated genes were determined using a one way ANOVA analysis and a Benjamini Hochberg False Discovery Rate method for Inhibitors,Modulators,Libraries multiple comparison corrections, followed by a Students post hoc test.
We performed a separate analysis that was designed to minimize the Inhibitors,Modulators,Libraries number of false negative genes. Those genes whose expression changed little these under the differ ent experimental conditions were filtered out. Specifi cally, only those genes whose expression changed at least 1. 3 fold from the baseline value in at least two of the hypoxia treated samples were selected for down stream analysis. This filter helped maximize the number of genes.