A total of 15 ovine fetuses were chronically catheterized: 8 were subjected to 30 min of maternal ventilatory hypoxia, and 7 were subjected to 30 min of normoxia. Within each condition, half of the fetuses were treated with ketamine (3 mg/kg), and half with vehicle (saline). Twenty-four hours following the onset of hypoxia or normoxia, the fetuses were euthanized and the kidney cortex were collected and snapped frozen.
Extracted molecule
total RNA
Extraction protocol
The RNA was extracted from the snapped frozen kidney cortex using Trizol (Invitrogen, Carlsbad, CA), then purified by using RNeasy Plus Mini kit (Qiagen, Valencia, CA) with RNase-Free DNase Set (Qiagen, Valencia, CA). The RNA concentration was measured using NanoDrop spectrophotometer (ND-1000, ThermoFisher, Wilmington, DE), and the quality was determined using 2100 Bioanalyzer (Agilent, Santa Clara, CA). The RNA Integrity Number (RIN) values of the samples ranged from 7.7 to 9.1.
Label
Cy3
Label protocol
Agilent Quick Amp Labeling Kit (5190-0442, Santa Clara, CA) was used to label 500 ng of RNA with Cyanine 3 (Cy3) CTP according to manufacturer’s protocol, except that the microcentrifugation of the samples were performed at room temperature instead of 4 °C. The labeled cRNA was analyzed with NanoDrop spectrophotometer, and the specific activities (10.4 to 12.7 pmol Cy3/μg RNA) and yields of the cRNAs were calculated (5.8 to 7.9 μg). The labeled cRNA was stored at -80 °C until further use.
Hybridization protocol
Hybridization was performed following Agilent protocol. 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 (019921, Agilent), 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 65 °C 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 Microarray Scanner System (G2505-90021, Agilent), and features were extracted with Agilent Feature Extraction 9.1 software at the Genomics Division of the University of Florida’s Interdisciplinary Center for Biotech Research. Features flagged as Feature Non-uniform outliers were excluded from further analysis.
Description
Gene expression 24 hrs after 30 min hypoxia in vehicle treated fetuses
Data processing
The limma package was used to import the raw data into R (http://www.r-project.org), performed background corrections, and normalized data by quantile normalization method. Probes that were at least 10% brighter than the negative controls on at least three arrays were retained for further analysis, and control probes and low expressed probes were filtered out.
