|
| Status |
Public on Dec 17, 2012 |
| Title |
Ssalar_2daypostSBMfeed_rep1_tank2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Distal intestine
|
| Organism |
Salmo salar |
| Characteristics |
day_post_soybeanmealfeed: 2
|
| Treatment protocol |
Day zero in this experiment marks the sampling of FM fed fish. Duplicate tanks of the remaining fish received the SBM test diet from day zero and were sampled after one, two, three, five and seven days of exposure.
|
| Growth protocol |
Atlantic salmon (Salmo salar L.) of the Sunndalsøra breed held in 1 m3 fiberglass tanks with running seawater (25-30 fish per tank) at the research facilities of Nofima Marin, Sunndalsøra, Norway. Initial body weight of 500-600 g. Two diets, the reference (FM) diet contained 563 g kg-1 fishmeal meal as the sole protein source while the test diet contained 200 g kg-1 hexane-extracted (defatted) soybean meal, partially replacing the fishmeal in the FM diet. Both diets were approximately iso-nitrogenous and iso-energetic on a crude protein and gross energy basis, containing 28% lipid and 43% crude protein. All fish were adapted to the FM diet for seven days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction, sections of distal intestinal tissue (distal-most region of the post-gastric intestinal tract defined by appearance of annular mucosal folds and widening of intestinal diameter) were taken and placed in RNAlater at 4°C for 24h and then stored at -80°C. To ensure exposure to SBM, only fish that had content throughout the intestinal tract were sampled. From ten fish per time point (five from each tank duplicate) approximately 20 mg of sample tissue was immersed in 1 ml TRIzol® (Invitrogen), homogenized with a mixermill (Retsch MM301) and total RNA was extracted as per manufacturers protocol. Total RNA was purified using RNeasy columns as per manufacturer’s instructions (Qiagen), including an optional on-column DNase I digestion step. Samples were quantified by spectrophotometry (NanoDrop1000™, Thermo Fisher Scientific) and quality checked by agarose gel electrophoresis. Samples confirmed free of genomic DNA by PCR with and without reverse transcriptase. Samples were stored at -80°C until further use.
|
| Label |
Cy5
|
| Label protocol |
Reverse transcription and labeling of the RNA was done according to Agilent’s two color Low Input Quick Amp labeling protocol (Version 6.5, May 2010).cRNA was quantified using spectrophotometry (NanoDrop1000™, Thermo Fisher Scientific). All samples had specific activity >6 pmol dye per µg cRNA. Labeled cRNA was stored at -80°C in the dark until hybridization.
|
| |
|
| Channel 2 |
| Source name |
Pooled 3 indiv intestine samples from each condition (n=18)
|
| Organism |
Salmo salar |
| Characteristics |
reference: Reference pool
|
| Treatment protocol |
Day zero in this experiment marks the sampling of FM fed fish. Duplicate tanks of the remaining fish received the SBM test diet from day zero and were sampled after one, two, three, five and seven days of exposure.
|
| Growth protocol |
Atlantic salmon (Salmo salar L.) of the Sunndalsøra breed held in 1 m3 fiberglass tanks with running seawater (25-30 fish per tank) at the research facilities of Nofima Marin, Sunndalsøra, Norway. Initial body weight of 500-600 g. Two diets, the reference (FM) diet contained 563 g kg-1 fishmeal meal as the sole protein source while the test diet contained 200 g kg-1 hexane-extracted (defatted) soybean meal, partially replacing the fishmeal in the FM diet. Both diets were approximately iso-nitrogenous and iso-energetic on a crude protein and gross energy basis, containing 28% lipid and 43% crude protein. All fish were adapted to the FM diet for seven days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction, sections of distal intestinal tissue (distal-most region of the post-gastric intestinal tract defined by appearance of annular mucosal folds and widening of intestinal diameter) were taken and placed in RNAlater at 4°C for 24h and then stored at -80°C. To ensure exposure to SBM, only fish that had content throughout the intestinal tract were sampled. From ten fish per time point (five from each tank duplicate) approximately 20 mg of sample tissue was immersed in 1 ml TRIzol® (Invitrogen), homogenized with a mixermill (Retsch MM301) and total RNA was extracted as per manufacturers protocol. Total RNA was purified using RNeasy columns as per manufacturer’s instructions (Qiagen), including an optional on-column DNase I digestion step. Samples were quantified by spectrophotometry (NanoDrop1000™, Thermo Fisher Scientific) and quality checked by agarose gel electrophoresis. Samples confirmed free of genomic DNA by PCR with and without reverse transcriptase. Samples were stored at -80°C until further use.
|
| Label |
Cy3
|
| Label protocol |
Reverse transcription and labeling of the RNA was done according to Agilent’s two color Low Input Quick Amp labeling protocol (Version 6.5, May 2010).cRNA was quantified using spectrophotometry (NanoDrop1000™, Thermo Fisher Scientific). All samples had specific activity >6 pmol dye per µg cRNA. Labeled cRNA was stored at -80°C in the dark until hybridization.
|
| |
|
| |
| Hybridization protocol |
Each experimental sample and reference pool (825 ng each) was fragmented for 30 minutes at 60°C, mixed with an equal amount of 2x GEx Hybridization buffer HI-RPM (Agilent Gene Expression Hybridization Kit, No. 5188-5242) and loaded onto a cGRASP 44k Salmonid oligonucleotide microarray (Agilent Technologies, [21]). The hybridization was incubated for 17 hrs (10 rpm at 65°C) in a hybridization oven (Agilent). Slides were washed immediately after hybridization using wash buffers, acetonitrile and Stabilization and Drying Solution, as per manufacturer’s instructions to prevent ozone-related problems. Slides were then kept in the dark at low ozone (<9 ppb) and scanned as soon as possible.
|
| Scan protocol |
All slides were scanned using a ScanArray® Express scanner (PerkinElmer®; 5μm resolution; PMTs: Cy5: 60, Cy3: 65) in a low-ozone environment (<9 ppb).
|
| Description |
Ssalar_2daypostSBMfeed_rep1_tank2
|
| Data processing |
Scanned images were processed in the ImaGene® software (V 8.0; Biodiscovery, El Segundo, CA, USA) using the 44K salmonid GAL file (11/09) (http://web.uvic.ca/grasp/microarray/array.html). The raw data was imported into GeneSpring GX11.5 (Agilent). Annotation files and specifications of the microarray can be found at the cGRASP microarray page (http://web.uvic.ca/grasp/microarray/array.html). All spots were thresholded to 1.0, samples were normalized using an intensity-dependent LOWESS normalization and a per gene baseline to median normalization. Control spots were removed and all entities were filtered to retain only those entities in which 80% of the biological replicates had raw values ≥500 in any one of the six conditions (i.e. time points). Entities were then filtered to retain the entities in which at least 80% were flagged as ‘present’ in six of six conditions. The remaining entities were tested for significant differential expression by comparing each of the time points (day one, two, three, five, seven) to day zero using the Mann-Whitney test (p<0.05) without multiple test correction. Only genes that changed more than 1.5 fold were considered for further analysis.
|
| |
|
| Submission date |
Apr 20, 2012 |
| Last update date |
Dec 18, 2012 |
| Contact name |
Ben F Koop |
| E-mail(s) |
bkoop@uvic.ca
|
| 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 |
GPL11299 |
| Series (1) |
| GSE37457 |
Early response of gene expression in the distal intestine of Atlantic salmon (Salmo salar L.) during the development of soybean meal induced enteritis |
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