|
| Status |
Public on Oct 30, 2010 |
| Title |
Liver Control - 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Liver from control male fathead minnow
|
| Organism |
Pimephales promelas |
| Characteristics |
gender: male
agent: control
tissue type: liver
|
| Biomaterial provider |
Fish were exposed by University of Minnesota. RNA was extracted at University of Florida.
|
| Treatment protocol |
Fathead minnow liver exposed in vivo for 3 weeks to clean water
|
| Growth protocol |
Adult (ca. 6 month old) male FHM were cultured at the University of Minnesota. Effluent was transported to the laboratory. Briefly, each day approximately 700 L (maximum trucking capacity) of effluent was pumped from the effluent channel into polypropylene containers (Nalgene) using a submersible pump. After being trucked to the lab, it was pumped using submersible pumps into a large fiberglass tank where it was heated to 25˚C (± 1˚C) using submersible heaters (Process Technologies, 1800 watts) before being pumped again into a head tank for delivery to exposure tanks. Exposures were performed as static renewal with effluent delivered at full strength (400 ml/min, for 4 hours) twice daily into exposure aquaria containing 3 females and 9 males (N=2 runs). Control tanks had the same number of fish but only received well water during the same 4 hour dosing periods. Fish exposure lasted 3 weeks. At the conclusion of the exposure, blood samples were collected for analysis of Vtg plasma concentrations. Liver and gonad samples were removed and stored in RNA later (Applied Biosystems/Ambion, Austin, TX). All procedures involving live fish were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated 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
|
| Organism |
Pimephales promelas |
| Characteristics |
reference: The reference pool consisted of equal amounts of RNA from control fathead minnow female and male tissues (liver, brain and gonad).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated 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 |
Microarray image processing and data pre-processing were performed using Agilent's Feature Extraction software v 9.5.
|
| Data processing |
Microarray image processing and data pre-processing were performed using Agilent's Feature Extraction software v 9.5.
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 |
Aug 06, 2010 |
| Last update date |
Aug 09, 2010 |
| 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) |
| GSE23490 |
Linkage of behavioral and genomic impacts of a wastewater effluent on the fathead minnow |
|
