|
| Status |
Public on Feb 20, 2008 |
| Title |
GRASP array Skin 24hr Post-Exposure Trout Lodge Rep4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Rainbow trout (Trout Lodge strain) skin exposed to M. cerebralis pathogen
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
Strain: Trout Lodge Age: 9 weeks old Tissue: Skin (caudal fin) Treatment: Exposed to 2,000 triactinomyxons per fish and tissue collected 24 hours after initial exposure.
|
| Biomaterial provider |
Melinda Baerwald
|
| Treatment protocol |
Each fish was exposed to 2,000 triactinomyxons (life stage of pathogen that infects salmonids) for one hour. Twenty four hours after initial exposure, fish was euthanized by overdose with benzocaine at 500 mg/L.
|
| Growth protocol |
Fish were reared in 15C flow-through well water for 9 weeks post-hatch.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from caudal fin (posterior to peduncle), comprised largely of skin tissue, was extracted using the ABI Prism TransPrep system with the ABI Prism 6100 Nucleic Acid PrepStation according to manufacturer’s instructions.
RNA quality was determined using agarose gel electrophoresis. RNA concentration was determine using a ND-1000 spectrophotometer (NanoDrop Technologies).
|
| Label |
Cy3
|
| Label protocol |
RNA (starting amount: 1000 ng) was amplified and indirect labeled according to the protocol provided with the Ambion Amino Allyl MessageAmp II aRNA Amplification kit.
|
| |
|
| Channel 2 |
| Source name |
Rainbow trout (Trout Lodge strain) skin unexposed control
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
Strain: Trout Lodge Age: 9 weeks old Tissue: Skin (caudal fin) Treated identically to exposed fish other than lack of pathogen exposure.
|
| Biomaterial provider |
Melinda Baerwald
|
| Treatment protocol |
No treatments other than euthanized by overdose with benzocaine at 500 mg/L.
|
| Growth protocol |
Fish were reared in 15C flow-through well water for 9 weeks post-hatch.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from caudal fin (posterior to peduncle), comprised largely of skin tissue, was extracted using the ABI Prism TransPrep system with the ABI Prism 6100 Nucleic Acid PrepStation according to manufacturer’s instructions.
RNA quality was determined using agarose gel electrophoresis. RNA concentration was determine using a ND-1000 spectrophotometer (NanoDrop Technologies).
|
| Label |
Cy5
|
| Label protocol |
RNA (starting amount: 1000 ng) was amplified and indirect labeled according to the protocol provided with the Ambion Amino Allyl MessageAmp II aRNA Amplification kit.
|
| |
|
| |
| Hybridization protocol |
Array Prewash:
1. 0.1% SDS for 5 minutes, repeat once
2. Filtered Milli-Q H20 (with DDT added) for 1 minute, repeat 4 times
3. Wash in near boiling water for 3 minutes
4. Immediately transfer to Sigma 4-15C centrifuge from Qiagen.
Centrifuge for 2 minutes at 1500 RPM to dry.
5. Wash Erie Scientific mSeries lifterslips (25X60) in filtered water and
dry with a Kimwipe to remove all traces of dust.
Microarray Prehybridization:
1. Incubate microarray for 90 minutes in 5XSSC, 0.1% SDS, 3% BSA
(Fraction V) at 49C.
2. Wash 3 time in Milli-Q H20 for 20 seconds
3. Dry slides immediately by centrifugation 514 x g for 5 minutes
4. Place the arrays into 49oC hybridization oven until hybridization
Microarray Hybridization:
1. Thaw 2X Formamide hybridization buffer in 55C water bath for 10
minutes. Ensure it’s thoroughly mixed by inversion.
2. Combine 6.5 ug of each labeled target (26 ul total) + 4 ul LNA dT
blocker + 30 ul of 2X formamide hybridization buffer.
3. Gently vortex in a microcentrifuge tube and briefly spin down.
4. Incubate at 80C for 10 minutes
5. Keep at 65C until loaded onto array
6. Place lifterslip on top of array. Slowly pipet hybridization solution
onto array at a corner of the lifterslip so that solution slowly
spreads out by capillary action.
7. Incubate overnight at 49C in a tightly sealed 50 ml conical tube
with 250 ul RNase-free water in bottom of tube. Wrap in foil to
ensure complete darkness.
Post-hybridization Washes:
Place arrays into staining trays. Perform all post-washes in foil-
wrapped staining dishes in a dark room.
1. Heat 2X SSC/0.1% SDS to 49C
2. Place array into dish (no more than 4 slides per dish) and gently
agitate tube for 10 minutes. Coverslip should float off.
3. Wash array (no coverslip) with 2X SSC/0.1% SDS (room temp) for
5 minutes, repeat once
4. Wash array 2X with 1X SSC (room temp)
5. Wash array 2X with 0.1X SSC (room temp)
6. Immediately transfer to Sigma 4-15C centrifuge from Qiagen.
Orient slide so array surface is facing out in the centrifuge, label is
down. Centrifuge for 2 minutes at 1500 RPM to dry.
7. Place slides in dark until ready to scan. Signal will decrease with
time so scan ASAP.
|
| Scan protocol |
Arrays were scanned using the Agilent G2565BA fluorescent scanner.
|
| Description |
NA
|
| Data processing |
Data were local background subtracted and normalized (LOWESS method) using Agilent's feature extraction software.
|
| |
|
| Submission date |
Jun 27, 2007 |
| Last update date |
Feb 20, 2008 |
| Contact name |
Melinda Baerwald |
| E-mail(s) |
mrbaerwald@ucdavis.edu
|
| Phone |
530-752-6351
|
| Organization name |
University of California, Davis
|
| Street address |
One Shields Ave
|
| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95833 |
| Country |
USA |
| |
|
| Platform ID |
GPL2899 |
| Series (1) |
| GSE8631 |
Discovery of genes associated with whirling disease infection and resistance in rainbow trout using expression profiling |
|
