|
| Status |
Public on Aug 21, 2007 |
| Title |
Head kidney,vaccine FO-7, pool 3B-rep4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from head kidney at 12 weeks post vaccination
|
| Organism |
Salmo salar |
| Characteristics |
Bolax strain
Fish vaccinated with M.viscosa (standard antigen concentration)
average side effect score: 2.05
average injection site inflammatory score=0.49
average weight(g)=103.0
|
| Treatment protocol |
Fish were divided into 4 groups of 50 each by dip netting and sequencial allocation. Two groups (100 fish) were kept in each tank. Marking was done by fin clipping under anaesthesia (0.5 ml/L Chlorobutanol (Sigma)). Each fish was intraperitoneally injected with 0.1 ml of experimental water-in-oil vaccines (Pharmaq, Norway).
|
| Growth protocol |
Atlantic salmon (Salmo salar) parr were reared in two 500 L tanks containing continuously running fresh water at an average temperature of 17 °C. Fish were fed with commercial dry pellets (EWOS, Norway).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNEasy® kit with on column DNAse treatment
|
| Label |
Cy3
|
| Label protocol |
2 μg of pooled total RNA (from 4 fish) were subjected to 1 round of amplification by using the Amino Allyl MessegeAmp II aRNA amplification kit (Ambion)
Coupling of aRNA was done with either Cy3 or Cy5 dye according to the Amino Ally MessageAMP™ II aRNA Amplification Kit (Ambion) protocol with minor modifications. Briefly, one vial of Cy3 or Cy5 reactive dyes (Amersham) was resuspended in 22 µl of DMSO prior to coupling. 3 µg of aRNA was dried to completion in a vacuum centrifuge (Savant Instruments, Inc., Farmingdale, NY). The aRNA was resuspended in 4.5 µl of coupling buffer. Thereafter, 5.5 µl of dye was added to the aRNA:coupling buffer and incubated for 30 min in the dark. 2.25 µl of 4M Hydroxylalamine was then added to quench the reaction followed by 12.75 µl of water to obtain a total volume to 30 µl. Dye labeled aRNA purification was done according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
Total RNA from head kidney at 12 weeks post vaccination
|
| Organism |
Salmo salar |
| Characteristics |
Bolax strain
Fish vaccinated with A. salmonicida (standard antigen concentration)
average side effect score= 0.15
average injection site inflammatory score=0.05
average weight(g)=109.7
|
| Treatment protocol |
Fish were divided into 4 groups of 50 each by dip netting and sequencial allocation. Two groups (100 fish) were kept in each tank. Marking was done by fin clipping under anaesthesia (0.5 ml/L Chlorobutanol (Sigma)). Each fish was intraperitoneally injected with 0.1 ml of experimental water-in-oil vaccines (Pharmaq, Norway).
|
| Growth protocol |
Atlantic salmon (Salmo salar) parr were reared in two 500 L tanks containing continuously running fresh water at an average temperature of 17 °C. Fish were fed with commercial dry pellets (EWOS, Norway).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNEasy® kit with on column DNAse treatment
|
| Label |
Cy5
|
| Label protocol |
2 μg of pooled total RNA (from 4 fish) were subjected to 1 round of amplification by using the Amino Allyl MessegeAmp II aRNA amplification kit (Ambion)
Coupling of aRNA was done with either Cy3 or Cy5 dye according to the Amino Ally MessageAMP™ II aRNA Amplification Kit (Ambion) protocol with minor modifications. Briefly, one vial of Cy3 or Cy5 reactive dyes (Amersham) was resuspended in 22 µl of DMSO prior to coupling. 3 µg of aRNA was dried to completion in a vacuum centrifuge (Savant Instruments, Inc., Farmingdale, NY). The aRNA was resuspended in 4.5 µl of coupling buffer. Thereafter, 5.5 µl of dye was added to the aRNA:coupling buffer and incubated for 30minutes in the dark. 2.25 µl of 4M Hydroxylalamine was then added to quench the reaction followed by 12.75 µl of water to obtain a total volume to 30 µl. Dye labeled aRNA purification was done according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
Post-print processing of arrays was done by washing 2 X 5 min in 0.2% SDS, 5 X 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.Prehybridization involved placing the arrays in (5 X SSC, 1% SDS and 3% BSA (Sigma)) and incubating at 49 °C in a water bath for 1½ hrs. Thereafter, the arrays were washed thrice for 20 sec in MilliQ water and dried by centrifugation as above. For hybridization, 500ng 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 16 hrs. 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 softwareScanning was done using ScanArray™ Express microarray skanner
|
| Description |
Head kidney,vaccine FO-7, pool 3B-rep4
|
| Data processing |
LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Genespring GX software was used.
|
| |
|
| Submission date |
Aug 20, 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) |
| GSE8826 |
High gene expression of inflammatory markers and IL-17A in Atlantic salmon vaccinated with oil-adjuvanted vaccines |
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