|
| Status |
Public on Mar 20, 2006 |
| Title |
3 dpa tcdd b |
| Sample type |
RNA |
| |
|
| Source name |
Adult Male Zebrafish Caudal Fin 3 Day Post Amputation, TCDD Exposed Replicate B
|
| Organism |
Danio rerio |
| Characteristics |
strain: AB Zebrafish
age: Adult
gender: Male
time point: 3 day post amputation of caudal fin
exposure: TCDD
|
| Treatment protocol |
Adult male zebrafish (Danio rerio) (AB strain, Eugene, OR) were anesthetized with tricaine methanesulfonate (MS-222) and their caudal fins were amputated at the bifurcation of the fin rays. Fish were then injected with phosphatidylcholine liposomes (vehicle control) alone or liposomes incorporated with TCDD (>99% pure, Chemsyn, Lenexa, KS) resulting in a final dose of 50 ng TCDD/g fish. At the indicated dpa, fish were euthanized with an over dose of MS-222. Regenerated fin tissue was then isolated by amputation within two ray segments anterior to the original amputation plane. Amputated tissue was then stored in RNAlater (Qiagen, Valencia, CA) and frozen at -80oC until RNA was isolated from the tissue. Tissues from 10 fish were combined to comprise a single experimental group (replicate). Three groups of tissue were obtained from vehicle control or TCDD exposed fish at 1, 3, and 5 dpa.
|
| Growth protocol |
Fish were matained at 27oC until under standard conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Amputated tissue was stored in RNAlater (Qiagen, Valencia, CA) and frozen at -80oC. RNAlater was removed from the samples and total RNA was purified with TRI reagent (Molecular Research Laboratories, Cincinnati, OH) according to the manufacturer’s instructions. Total RNA was DNase treated with RQ1 DNase (Promega, Madison, WI) according to the manufacture’s protocol and RNA quantity and quality was determined by UV absorbance. Ribosomal RNA abundance and degree of degradation was determined in electropherogram patterns using the 2100 Bioanalyzer and RNA 6000 Nano chips (Agilent Technologies, Palo Alto, CA).
|
| Label |
Labeling and probe processing was conducted under manufactures recommendations.
|
| Label protocol |
2.5 µg of total RNA was used to generate biotinylated complementary RNA (cRNA) for each treatment group using the One-Cycle Target Labeling kit (Affymetrix, Santa Clara, CA). RNA was reverse transcribed using a T7-(dT)24 primer and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA).
|
| |
|
| Hybridization protocol |
Hybridization was conducted as stipulated by Affymetrix GeneChip Expression Analysis Technical Manual (701021 Rev. 5).
|
| Scan protocol |
Affymetrix fluidics station 400 was used to wash the arrays. Arrays were scanned with an Affymetrix scanner 3000.
|
| Description |
Adult male zebrafish were i.p. injected with 50 ng/g TCDD or vehicle and caudal fins were amputated. Regenerating fin tissue was collected at 1, 3 and 5 days post amputation for mRNA abundance analysis.
|
| Data processing |
Data was GC-RMA preprocessed and each gene was normalized to the median using Gene Spring 7.1 software.
|
| |
|
| Submission date |
Nov 23, 2005 |
| Last update date |
Jun 13, 2011 |
| Contact name |
Robert L Tanguay |
| E-mail(s) |
robert.tanguay@oregonstate.edu
|
| Phone |
541-737-6514
|
| Organization name |
Oregon State University
|
| Department |
EMT
|
| Street address |
1007 ALS
|
| City |
Corvallis |
| State/province |
OR |
| ZIP/Postal code |
97331 |
| Country |
USA |
| |
|
| Platform ID |
GPL1319 |
| Series (1) |
| GSE3667 |
1, 3, or 5 Day Post Amputation Vehicle or TCDD Exposed |
|
