developmental stage: fetal stress: Control tissue: hypothalamus
Treatment protocol
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.
Extracted molecule
total RNA
Extraction protocol
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.
Label
Cy3
Label protocol
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 Agilent’s 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/µg RNA and from 4.29 to 10.38 µg, respectively. The labeled cRNA was stored at -80oC until use.
Hybridization protocol
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.
Scan protocol
The arrays were hybridized, washed, dried, stabilized, and scanned at 5 μm at both 10 and 100 PMT with an Agilent G2505B Microarray scanner at the Interdisciplinary Center for Biotechnology Research at the University of Florida.
Description
con_1 Array 7
Data processing
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).
