|
| Status |
Public on Nov 15, 2007 |
| Title |
AGD lesion fish 4 (IF6L) vs Control fish 5 (CF9) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from AGD-affected gill lesion tissue at 36 days post inoculation with Neoparamoeba perurans
|
| Organism |
Salmo salar |
| Characteristics |
AGD-affected fish 4, AGD lesion tissue, IF6L
Dissected lesion tissue from AGD-affected gill arch
Fish inoculated with Neoparamoeba perurans, the aetiological agent of AGD
weight: 79.5
|
| Treatment protocol |
Amoebae, later confirmed to be Neoparamoeba perurans were harvested from the gills of moribound AGD-affected Atlantic salmon (Salmo salar). Twenty fish in one 500L tank were exposed to N. perurans at 500 cells L-1 while the other tank (20 fish) became the control AGD-naive group. Fish were held for 36 days then fish were euthanised and the gill filaments of fish in both treatments were excised and stored in RNAlater (Sigma-Aldrich).
|
| Growth protocol |
Forty Atlantic salmon (Salmo salar) were reared in two 500L tanks (20 fish per tank). Fish were acclimated from freshwater to seawater (35 ppt) over a three week period then maintained in recirculated seawater at an average temperature of 15.5°C . Fish were fed with commercial pellets (Skretting, Australia).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNEasy® kit (Qiagen) with on-column DNAse treatment
|
| Label |
Cy3
|
| Label protocol |
2μg of total RNA from each fish was subjected to 1 round of amplification by using the Amino Allyl MessageAmp I aRNA amplification kit (Ambion)
Coupling of aRNA was performed using either Cy3 or Cy5 dye according to the Amino Ally MessageAMP™ I aRNA Amplification Kit (Ambion) protocol with minor modifications. Briefly, one vial of Cy3 or Cy5 reactive dyes (Amersham) was resuspended in 22µl of DMSO prior to coupling. 3µg of aRNA was dried to completion in a vacuum centrifuge (Savant Instruments, Inc., Farmingdale, NY). The aRNA was resuspended in 4.5µl of coupling buffer. Thereafter, 5.5µl of dye was added to the aRNA:coupling buffer and incubated for 30 minutes in the dark. 2.25µl of 4M hydroxylalamine was then added to quench the reaction followed by 12.75µl of water to obtain a total volume to 30µl. Dye-labeled aRNA purification was performed according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
Total RNA from AGD-naive gill non-lesion tissue
|
| Organism |
Salmo salar |
| Characteristics |
AGD-naive fish 5, no lesion tissue, CF9
Dissected non-lesion tissue from AGD-naive gill arch
Fish inoculated with Neoparamoeba perurans, the aetiological agent of AGD
Weight: 85.3
|
| Treatment protocol |
Amoebae, later confirmed to be Neoparamoeba perurans were harvested from the gills of moribound AGD-affected Atlantic salmon (Salmo salar). Twenty fish in one 500L tank were exposed to N. perurans at 500 cells L-1 while the other tank (20 fish) became the control AGD-naive group. Fish were held for 36 days then fish were euthanised and the gill filaments of fish in both treatments were excised and stored in RNAlater (Sigma-Aldrich).
|
| Growth protocol |
Forty Atlantic salmon (Salmo salar) were reared in two 500L tanks (20 fish per tank). Fish were acclimated from freshwater to seawater (35 ppt) over a three week period then maintained in recirculated seawater at an average temperature of 15.5°C . Fish were fed with commercial pellets (Skretting, Australia).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNEasy® kit (Qiagen) with on-column DNAse treatment
|
| Label |
Cy5
|
| Label protocol |
2μg of total RNA from each fish was subjected to 1 round of amplification by using the Amino Allyl MessageAmp I aRNA amplification kit (Ambion)
Coupling of aRNA was performed using either Cy3 or Cy5 dye according to the Amino Ally MessageAMP™ I aRNA Amplification Kit (Ambion) protocol with minor modifications. Briefly, one vial of Cy3 or Cy5 reactive dyes (Amersham) was resuspended in 22µl of DMSO prior to coupling. 3µg of aRNA was dried to completion in a vacuum centrifuge (Savant Instruments, Inc., Farmingdale, NY). The aRNA was resuspended in 4.5µl of coupling buffer. Thereafter, 5.5µl of dye was added to the aRNA:coupling buffer and incubated for 30 minutes in the dark. 2.25µl of 4M hydroxylalamine was then added to quench the reaction followed by 12.75µl of water to obtain a total volume to 30µl. Dye-labeled aRNA purification was performed according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
Post-print processing of arrays was done by washing 2X 5 min in 0.2% SDS, 5X 1 min in MilliQ water followed by immersion in MilliQ water at 100°C for 3 min. The slides were dried by centrifugation at 512 X g for 5 min.Pre-hybridization involved placing the arrays in (5X SSC, 1% SDS and 3% BSA (Sigma)) and incubating at 49°C in a water bath for 90 min. Thereafter, the arrays were washed thrice for 20 sec in MilliQ water and dried by centrifugation as above. For hybridization, 500 ng of labeled target reconstituted to a final volume of 26 µL, 30 µL of 2X formamide hybridization buffer (Genisphere) and 4 µL LNT dT blocker (Genisphere) were used. Hybridization was done at 49°C in a water bath for 16hrs. The arrays were washed first in (2X SSC, 0.1% SDS) for 10 min at 49°C, 2 X 5 min in (2X SSC, 0.1%SDS) at room temperature, 2 X 5 min in 1X SSC, 4 X 5 min in 0.1X SSC followed immediately by centrifugation at 512 X g for 5 min.
|
| Scan protocol |
Imaging was done at 10 µm resolution using ScanArray™ Express microarray scanner (Packard Bioscience). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm, respectively, at the same laser power (90%), with adjusted photomultiplier tube settings between slides to balance the Cy5 and Cy3 channels. Image analysis and fluorescent intensity data was extracted from Tiff images using ImaGene™ 5.6 Standard Edition software. Scanning was done using ScanArray™ Express microarray scanner.
|
| Description |
AGD lesion fish 4 (IF6L) vs Control fish 5 (CF9)
|
| Data processing |
LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Genespring GX software was used.
|
| |
|
| Submission date |
Nov 13, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Ben F Koop |
| E-mail(s) |
[email protected]
|
| Phone |
(250) 472-4067
|
| Organization name |
The University of Victoria
|
| Department |
Biology
|
| Lab |
Centre for Biomedical Research
|
| Street address |
PO Box 3020 STN CSC
|
| City |
Victoria |
| State/province |
BC |
| ZIP/Postal code |
V8W 3N5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL2716 |
| Series (1) |
| GSE9595 |
Global gene expresssion profiling the gills of amoebic gill disease (AGD) affected Atlantic salmon (Salmo salar) |
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