|
| Status |
Public on May 13, 2009 |
| Title |
FAD in vitro - 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Fathead minnows ovary exposed to fadrozole
|
| Organism |
Pimephales promelas |
| Characteristics |
tissue: ovary
|
| Biomaterial provider |
Fish were exposed at EPA (Duluth). RNA was extracted at University of Florida.
|
| Treatment protocol |
Fathead minnow ovary exposed in vitro for 12h to fadrozole, or solvent control
|
| Growth protocol |
Ovaries from 6 replicate fish were sliced into 12±5 mg pieces and randomly distributed across sample culture wells to minimize sample effects due to potential tissue heterogeneity. Ovary slices were incubated in tissue culture medium supplemented with IBMX (0.1 mM), 25-hydroxycholesterol (1ug/ml), and 50 uM FAD, or solvent control (0.07% methanol) for intervals of 1 to 12 hrs. Replicate ovary slices were removed at appropriate times, snap frozen in liquid nitrogen, and stored at -80°C until needed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from gonadal tissue with the RNA Stat-60 reagent (Tel-test, Friendswood, TX).
|
| Label |
Cy5
|
| Label protocol |
cDNA synthesis, cRNA labelling, amplification and hybridization were performed following the manufacturer's kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit)
|
| |
|
| Channel 2 |
| Source name |
Reference sample
|
| Organism |
Pimephales promelas |
| Characteristics |
sample type: liver, brain, and gonad pool
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from gonadal tissue with the RNA Stat-60 reagent (Tel-test, Friendswood, TX).
|
| Label |
CY3
|
| Label protocol |
cDNA synthesis, cRNA labelling, amplification and hybridization were performed following the manufacturer's kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit)
|
| |
|
| |
| Hybridization protocol |
Standard Agilent two-color protocol (Agilent 60-mer oligo microarray processing protocol)
|
| Scan protocol |
The microarrays were washed and scanned with a laser-based detection system (Agilent, Palo Alto, CA)
|
| Description |
RNA from fathead minnow ovary exposed in vitro to a 50 ug/L fadrozole
The reference pool consisted of equal amounts of RNA from control fathead minnow female and male tissues (liver, brain and gonad).
Microarray image processing and data pre-processing were performed using Agilent's Feature Extraction software v 9.5.
|
| Data processing |
The intensity of each spot was summarized by the median pixel intensity. A log2 transformed signal ratio between the experimental (Cy5) channel and the reference (Cy3) channel was calculated for each spot, followed by within-array lowess transformation and between array scale normalization on median intensities (Zahurak et al., 2007). Probes that did not hybridize were removed from consideration.
|
| |
|
| Submission date |
Apr 30, 2009 |
| Last update date |
May 12, 2009 |
| Contact name |
Natalia Vinas |
| E-mail(s) |
[email protected], [email protected]
|
| Phone |
6016343764
|
| Organization name |
Mississippi State University
|
| Street address |
3909 Halls Ferry Rd
|
| City |
Vicksburg |
| State/province |
MS |
| ZIP/Postal code |
39180 |
| Country |
USA |
| |
|
| Platform ID |
GPL7282 |
| Series (1) |
| GSE15924 |
Fathead minnow ovaries exposed in vivo or in vitro to fadrozole |
|
