|
| Status |
Public on Jan 03, 2009 |
| Title |
MIX (50-100) - 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Fathead minnows testis exposed to a mix of EE2 and ZM189,154
|
| Organism |
Pimephales promelas |
| Characteristics |
RNA from fathead minnow testis exposed to a mix of 50 ng/L ethinylestradiol and 100 ng/L ZM189,154
|
| Biomaterial provider |
Fish were exposed at University of Florida. RNA was extracted at University of Florida.
|
| Treatment protocol |
Fathead minnow ovary exposed for 48h to EE2, ZM189,154, a mix of EE2/ZM, or control (non-vehicle control or 70% triethylene glycol, TEG)
|
| Growth protocol |
Reproductively-mature, pond-reared FHM were purchased from Andersen Minnow Farm, AR, 4 days prior to starting the experiment. Upon arrival, the fish were treated for parasites and bacteria by a prophylactic salt-water dip (3%, 1 min). Males were separated from the population the following day, and acclimated in the treatment aquaria for 48 h. The water used for this study was carbon-filtered, dechlorinated tap water. The exposure system consisted of 40 L glass aquaria. Each exposure was conducted in quadruplicate and each aquarium contained eight male fathead minnows in 25 L of treatment water. Test chemicals for each treatment group (100L for 4 aquaria) were prepared in separate (by treatment) 250 L fiberglass tanks the day of exposure. Aquaria were equilibrated with test chemicals for 24 h prior to the introduction of fish. Test solutions were renewed to 90% of the 25L exposure volume after 24 h and the exposure was ended at 48 h. The positions of the treatment tanks were randomized and test initiation times were staggered to ensure an exposure/sampling interval of 48 h. The fish were not fed the day before and during the experiment. Temperature was maintained at 25oC with a photoperiod of 16 h light: 8 h dark.
|
| 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 Pool
|
| Organism |
Pimephales promelas |
| Characteristics |
The reference pool consisted of equal amounts of RNA from control fathead minnow female and male tissues (liver, brain and gonad).
|
| Biomaterial provider |
University of Florida
|
| 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 |
Microarrays were scanned with a laser-based detection system (Agilent, Palo Alto, CA).
|
| Description |
Four replicates consisting of four different individuals were analyzed for each of the treatments (solvent (TEG) control, non-solvent control, EE2, ZM, EE2 /ZM). The cDNA synthesis, cRNA labeling and hybridization were performed following the manufacturer’s kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA). The gonad samples were labeled with Cy5 while the reference sample was labeled with Cy3. Once the labeling was complete, samples were hybridized to the microarray using conditions recommended by the manufacturer. After hybridizing for 17 h, microarrays were washed and then scanned with a laser-based detection system (Agilent, Palo Alto, CA).
|
| 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 log10 transformed signal ratio between the experimental (Cy5) channel and the reference (Cy3) channel was calculated for each spot, followed by within-array lowess transformation.
|
| |
|
| Submission date |
Dec 29, 2008 |
| Last update date |
Jan 02, 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 |
GPL6516 |
| Series (1) |
| GSE14235 |
Gene expression signatures for 17α-ethinylestradiol and ZM 189,154, singly and combined, in fathead minnow testis |
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