|
| Status |
Public on Oct 01, 2015 |
| Title |
Normoxia_Replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Fetal Pituitary, Normoxia
|
| Organism |
Ovis aries |
| Characteristics |
tissue: Fetal Pituitary
developmental age: 128
Sex: male
treatment: normoxia
|
| Treatment protocol |
Chronically catheterized late gestation fetal sheep were subjected to 30 min hypoxia (n=4) or normoxia (n=4). Fetuses were euthanized 60 min after the onset of hypoxia or normoxia and pituitaries removed for microarray analysis. Gene expression was measured using Agilent 15k ovine microarrays.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from fetal hyothalamus with Trizol (Life Technologies, Carlsbad, CA), and purified through Qiagen RNeasy+ kits with on-column DNase digestion (QIAGEN, Valencia, CA) according to manufacturers' protocols.Total RNA concentration was measured by Nanodrop ND-1000 and RNA quality was monitored by Agilent Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 500 ng RNA using the One-Color Quick Amp Labeling Kit (Agilent, Newcastle DE) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity: 10.2 to 12.8 pmol Cy3/μg RNA) was fragmented and hybridized to Agilent Sheep Oligo Microarrays (G4813A) design 019921 by the ICBR facility at the University of Florida according to Agilent's methodology.
|
| Scan protocol |
Slides were scanned on the Agilent DNA Microarray Scanner (G2505B) using one color (green) scan setting for 8x15k array slides, by the ICBR facility at University of Florida.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.1.1.1 (Agilent) using default parameters (protocol GE1-v5_10 and Grid: 019921_D_F_20100112) background detrended Processed Signal. The limma package was employed to import the raw data into R (http://www.r-project.org), perform background correction and normalize the data by the quantile normalization method. Control probes and low expressed probes were filtered out, retaining for further analysis the probes that were at least 10% brighter than the negative controls on at least four arrays.
|
| |
|
| Submission date |
May 26, 2015 |
| Last update date |
Oct 01, 2015 |
| Contact name |
Charles Evans Wood |
| E-mail(s) |
woodc@ufl.edu
|
| Phone |
352-294-5064
|
| Organization name |
University of Florida
|
| Department |
Physiology and Functional Genomics
|
| Street address |
1345 Center Drive/Room M552
|
| City |
GAINESVILLE |
| State/province |
Florida |
| ZIP/Postal code |
32610-0274 |
| Country |
USA |
| |
|
| Platform ID |
GPL14112 |
| Series (1) |
| GSE69246 |
Transcriptomics Modeling of the Late-Gestation Fetal Pituitary Response to Transient Hypoxia |
|
