|
| Status |
Public on Mar 22, 2007 |
| Title |
Gonad Fish1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Gonad Fish1
|
| Organism |
Danio rerio |
| Characteristics |
Gonad Tissue
|
| Growth protocol |
Gonads extracted from adult breeding zebrafish kept in 15L breeding tanks ( 10 L of water were displaced daily), inder a 12 light/dark cycle, and fed twice daily with commercial flake food and live Artemia.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the TRI reagent method (Sigma, UK) followed by a LiCl precipitation. A RNA amplification step was carried out using the Amino Allyl MessageAmp aRNA amplification kit (Ambion, USA).
|
| Label |
Cy3
|
| Label protocol |
5ug of aaRNA were labelled with Cy3 using FluoroLink Cy3 monofunctional dye 5-pack (Amersham), according to the instructions provided within the Amino Allyl MessageAmp aRNA amplification kit (Ambion, USA).
|
| |
|
| Channel 2 |
| Source name |
Pool of Danio rerio gonadal RNA
|
| Organism |
Danio rerio |
| Characteristics |
Pool of 5ug of aRNA from each gonadal extract included in this experiment
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the TRI reagent method (Sigma, UK) followed by a LiCl precipitation. A RNA amplification step was carried out using the Amino Allyl MessageAmp aRNA amplification kit (Ambion, USA).
|
| Label |
Cy5
|
| Label protocol |
5ug of aaRNA Pool was labeled with Cy5 using FluoroLink Cy5 monofunctional dye 5-pack (Amersham), according to the instructions provided within the Amino Allyl MessageAmp aRNA amplification kit (Ambion, USA).
|
| |
|
| |
| Hybridization protocol |
The reference and sample, labeled with Cy5 and Cy3, respectively, were combined and denatured for 3 minutes at 95oC. An equal volume of hybridization buffer (50% formamide, 10X SSC, 0.2% SDS) was added to each sample, and the mixture was added to the arrayed slides. The slides were hybridized in a humidified airtight box for 36 hours at 42oC.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 with a Array Express fluorescent scanner and image intensity data were extracted and analyzed with Imagene V5 analysis software.
|
| Description |
Gonad Fish1
|
| Data processing |
Pre-processed in R using the LimmaGUI package with background subtraction, followed by a pin based-Lowess within array normalization and a scaled between chip normalization. For each experimental group, data were filtered on ‘flags’ and on intensity of the raw data, and each spot was required to have flag=1 and above 0.01 in at least 40% of the conditions to be considered for further analysis
|
| |
|
| Submission date |
Oct 02, 2006 |
| Last update date |
Mar 22, 2007 |
| Contact name |
Eduarda M Santos |
| E-mail(s) |
e.santos@exeter.ac.uk
|
| Phone |
00 44 1392 263752
|
| Fax |
00 44 1392 263700
|
| Organization name |
University of Exeter
|
| Department |
School of Biosciences
|
| Lab |
Environmental and Molecular Fish Biology
|
| Street address |
Prince of Wales Road
|
| City |
Exeter |
| State/province |
Devon |
| ZIP/Postal code |
EX4 4PS |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL4375 |
| Series (1) |
| GSE6063 |
Molecular basis of gender and reproductive status in breeding zebrafish |
|
