|
| Status |
Public on Aug 15, 2013 |
| Title |
Fetus from pregnant ewe exposed to 110 days of hypoxia at high altitude 1 |
| Sample type |
RNA |
| |
|
| Source name |
Carotid Arteries, hypoxia
|
| Organism |
Ovis aries |
| Characteristics |
maternal treatment: hypoxia
gestation: 140d
tissue: fetal carotid arteries
|
| Treatment protocol |
For the hypoxia study, at 30 days gestation, ewes were transported to the Barcroft Laboratory, White Mountain Research Station (WMRS, CA; elevation 3,801 m, barometric pressure -480 Torr), where they were kept until 135 days gestation. They were maintained in an sheltered outdoor pen that was sheltered and were fed with alfalfa pellets ad libitum. Sheep in both groups were kept in natural day-night conditions.
For control (normoxic) group, sheep were maintained at the suppliers ranch (a 300 m elevation) on alfalfa pellets ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue samples were lysed in Tri-reagent (Ambion, Austin, TX) and total RNA was isolated using phenol/chloroform extraction, followed by purification over spin columns (Ambion). The concentration and purity of total RNA was measured by spectrophotometry at OD260/280, and the quality of the total RNA sample was assessed using an Agilent Bioanalyzer with the RNA6000 Nano Lab Chip (Agilent Technologies).
|
| Label |
cy3
|
| Label protocol |
Labeled cRNA was prepared by linear amplification of the Poly(A)+ RNA population within the total RNA sample. Briefly, 1 µg of total RNA was reverse transcribed after priming with a DNA oligonucleotide containing the T7 RNA polymerase promoter 5’ to a d(T)24 sequence. After second-strand cDNA synthesis and purification of double-stranded cDNA, in vitro transcription was performed using T7 RNA polymerase. The quantity and quality of the labeled cRNA was assayed by spectrophotometry and the Agilent Bioanalyzer.
|
| |
|
| Hybridization protocol |
One µg of purified cRNA was fragmented to uniform size and applied to Agilent Sheep Gene Expression Microarray, 8 x 15K (Design ID 019921, Agilent Technologies) in hybridization buffer. Arrays were hybridized at 65° C for 17 hrs. in a shaking incubator and washed at 37° C for 1 min.
|
| Scan protocol |
Rinsed and dried arrays were scanned with an Agilent G2565 Microarray Scanner (Agilent Technologies) at 5 µm resolution.
|
| Description |
Transcriptome analysis
|
| Data processing |
Agilent Feature Extraction software was used to process the scanned images from arrays (gridding and feature intensity extraction) and the data generated for each probe on the array was analyzed with Gene Spring GX v7.3.1 software (Agilent Technologies). Annotations are based on the Agilent eArray annotation file dated January, 2010.
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| |
|
| Submission date |
Aug 15, 2013 |
| Last update date |
Aug 15, 2013 |
| Contact name |
Ravi Goyal |
| E-mail(s) |
rgoyal@live.com
|
| Phone |
9092531596
|
| Organization name |
University of Arizona
|
| Street address |
1501 N. Campbell Ave
|
| City |
TUCSON |
| State/province |
Arizona |
| ZIP/Postal code |
85724 |
| Country |
USA |
| |
|
| Platform ID |
GPL10778 |
| Series (1) |
| GSE49920 |
Antenatal maternal long-term hypoxia: acclimatization responses with altered gene expression in ovine fetal carotid arteries |
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