Comment[ArrayExpressAccession] E-GEOD-43371 MAGE-TAB Version 1.1 Public Release Date 2014-01-16 Investigation Title The Genomics of the Fetal Hypothalamic Cellular Response to Transient Hypoxia: Endocrine, Immune, and Metabolic Responses Comment[Submitted Name] The Genomics of the Fetal Hypothalamic Cellular Response to Transient Hypoxia: Endocrine, Immune, and Metabolic Responses Experiment Description Fetuses respond to transient hypoxia (a common stressor in utero) with cellular responses that are appropriate for promoting survival of the fetus. The present experiment was performed to identify the acute genomic responses of the fetal hypothalamus to transient hypoxia. Three fetal sheep were exposed to 30 min of hypoxia and hypothalamic mRNA extracted from samples collected 30 min after return to normoxia. These samples were compared to those from 4 normoxic control fetuses using the Agilent 019921 ovine array. Differentially-regulated genes were analyzed by network analysis and by gene ontology analysis, identifying statistically significant overrepresentation of biological processes. Real-time PCR of selected genes supported the validity of the array data. Hypoxia induced increased expression of genes involved in response to oxygen stimulus, RNA splicing, anti-apoptosis, vascular smooth muscle proliferation, and positive regulation of Notch receptor target. Downregulated genes were involved in metabolism, antigen receptor-mediated immunity, macromolecular complex assembly, S-phase, translation elongation, RNA splicing, protein transport, and post-transcriptional regulation. We conclude that these results emphasize that the cellular response to hypoxia involves reduced metabolism, the involvement of the fetal immune system, and the importance of glucocorticoid signaling. 3 Ventilatory Hypoxia (VH) and 4 Control (con) fetuses. All fetuses were chronically catheterized and in late gestation. Hypoxia produced by low PO2 in maternal inspired gas for 30 min, followed by normoxia recovery for 30 min. Control fetuses maintained at normoxia for 30 min, followed by another 30 min of normoxia. Hypothalami collected for mRNA at end of normoxic recovery period. 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 Wood Wood Rabaglino Chang Denslow Keller-Wood Richards Person First Name Charles Charles Maria Eileen Nancy Maureen Elaine Person Mid Initials E B Person Email woodc@ufl.edu Person Affiliation University of Florida Person Phone 352-294-5064 Person Address Physiology and Functional Genomics, University of Florida, 1345 Center Drive/Room M552, GAINESVILLE, Florida, USA Person Roles submitter Protocol Name P-GSE43371-1 P-GSE43371-4 P-GSE43371-5 P-GSE43371-2 P-GSE43371-3 P-GSE43371-6 Protocol Description Features were extracted with Agilent Feature extraction 9.1 software. Features flagged as Feature Non-uniform outliers were excluded from further analysis. Transcript levels were normalized to the chip median and log-transformed, in order to obtain more power in discovering differences between groups and compensate for systematic differences between the arrays. To identify the genes that were differentially regulated (DR) between the treated and control fetuses, the normalized and transformed intensities were analyzed by one-way ANOVA (p<0.01). All the statistical procedures were carried out using JMP Genomics 5 software (SAS Institute Inc, Cary, NC, USA). ID_REF = VALUE = normalized and log-transformed intensities Five hundred ng of the DNase-treated RNA was labeled with Cyanine 3 (Cy3) CTP with the Agilent Quick Amp kit (5190-0442, New Castle, DE) according to their methodology, purified with the Qiagen RNeasy kit (Valencia, CA) according to AgilentM-bM-^@M-^Ys revision of the Qiagen protocol as shown in the Quick Amp kit protocol except that the microcentrifugation was performed at room temperature instead of 4oC. The resulting labeled cRNA was analyzed with the NanoDrop spectrophotometer, and the specific activities and the yields of the cRNAs were calculated; these ranged from 9.0 to 12.8 pmol Cy3/M-5g RNA and from 4.29 to 10.38 M-5g, respectively. The labeled cRNA was stored at -80oC until use. This was performed following protocols from Agilent. Briefly 600 ng of each labeled cRNA was fragmented and then mixed with hybridization buffer using the Agilent gene expression hybridization kit. These were applied to a sheep 8 X 15 K array slide (Agilent 019921), containing 8 arrays with 15,744 oligomers with a length of 60 bases corresponding to 15,208 ovine genetic sequences published in the NCBI data base plus 500+ quality control probes provided by Agilent and hybridized at 65oC for 17 h at 10 rpm. Chronically catheterized late gestation fetal sheep were subjected to transient (30 min) maternal ventilatory hypoxia (n=3) or normoxia (n=4) followed by 30 min normoxia. Hypothalami were collected at the end of normoxia recovery period, and compared to hypothalami from age-matched controls not subjected to hypoxia. RNA was extracted from the hypothalamus using Trizol (Invitrogen, Carlsbad, CA) , followed by RNeasy+ kits with on-column DNase treatment (Qiazol, Valencia, CA).The RNA concentration was determined with a Nanodrop spectrophotometer (ND-1000, ThermoFisher, Wilmington DE) and the integrity of the RNA was measured using an Agilent Bioanalyzer, 2100 model. RNA Integrity Number (RIN) values ranged from 7.4 to 8.4. The arrays were hybridized, washed, dried, stabilized, and scanned at 5 M-NM-