|
| Status |
Public on Feb 24, 2014 |
| Title |
Chm_AK_d6_con_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Chum_salmon-anterior_kidney
|
| Organism |
Oncorhynchus keta |
| Characteristics |
exposure: con
day: 6
|
| Treatment protocol |
Ten size-matched (approx. 55-70g) individuals from each species were randomly allocated to each of eight tanks. After seven days acclimation, 5014 copepodids (167/fish) were added to each of four tanks using the metomidate hydrochloride (Aquacalm) sedation exposure method described previously (Jones et al. 2006). Two unexposed tanks were treated the same except with the addition of seawater without copepodids. At three, six, nine and 12 dpe, salmon were sampled by initial sedation with 0.5 mg/L metomidate, then a lethal immersion in 200 mg/L MS-222 followed by blunt head trauma, as previously described (Jones et al., 2007). The left pectoral fin and the anterior kidney were rapidly extracted from each fish and placed in liquid nitrogen, then stored at -80 °C until RNA extraction.
|
| Growth protocol |
Pink and chum salmon were obtained as swim-up fry from the Quinsam River and Nanaimo River hatcheries, respectively, on Vancouver Island, British Columbia. Atlantic salmon were obtained as parr from a commercial hatchery on Vancouver Island. The fish were reared in 400 L tanks in flowing water that was an equal mixture of aerated freshwater and seawater and were fed a diet of commercial salmon pellets at a daily rate of 1.0% biomass. Seawater was at 10 degrees Celcius and 30‰ salinity Ovigerous Lepeophtheirus salmonis were collected from adult Atlantic salmon following harvest from a farm near Vancouver Island and transported in ice cold aerated seawater to Nanaimo. Dissected egg strings were incubated in filtered and ultraviolet irradiated seawater at 9.5 ± 1.0 C and 29.5 ± 0.5‰ salinity, with supplemental aeration, as described previously (Jones et al. 2006). Cultured lice were monitored by daily microscopic examination of triplicate samples and an inoculum containing a known number of copepodids was prepared when the ratio of copepodid to nauplius II stages was greatest.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from fin and kidney samples using Trizol, as per manufacturer’s instructions, and purified using RNeasy spin columns (QIAGEN), by manufacturer’s instructions with the on-column DNase I digestion. The RNA was quality checked by agarose gel electrophoresis and quantified by spectrometry (NanoDrop-1000).
|
| Label |
Cy5-CTP
|
| Label protocol |
Total RNA samples from Trial 3 were randomized and 200ng total RNA of each was reverse transcribed to cDNA and amplified to labelled-cRNA using Low Input Quick Amp labelling kits as per manufacturer’s instructions (Agilent). Reference channel Cy3-cRNA was constructed by combining equimolar cRNA proportions from each species/infection class conditions, and from each condition including at least one representative from each day (n = 19).
|
| |
|
| Channel 2 |
| Source name |
pool of samples from all experimental conditions, containing equimolar sample(s) per condition
|
| Organism |
Oncorhynchus keta |
| Characteristics |
tag: Reference pool
|
| Treatment protocol |
Ten size-matched (approx. 55-70g) individuals from each species were randomly allocated to each of eight tanks. After seven days acclimation, 5014 copepodids (167/fish) were added to each of four tanks using the metomidate hydrochloride (Aquacalm) sedation exposure method described previously (Jones et al. 2006). Two unexposed tanks were treated the same except with the addition of seawater without copepodids. At three, six, nine and 12 dpe, salmon were sampled by initial sedation with 0.5 mg/L metomidate, then a lethal immersion in 200 mg/L MS-222 followed by blunt head trauma, as previously described (Jones et al., 2007). The left pectoral fin and the anterior kidney were rapidly extracted from each fish and placed in liquid nitrogen, then stored at -80 °C until RNA extraction.
|
| Growth protocol |
Pink and chum salmon were obtained as swim-up fry from the Quinsam River and Nanaimo River hatcheries, respectively, on Vancouver Island, British Columbia. Atlantic salmon were obtained as parr from a commercial hatchery on Vancouver Island. The fish were reared in 400 L tanks in flowing water that was an equal mixture of aerated freshwater and seawater and were fed a diet of commercial salmon pellets at a daily rate of 1.0% biomass. Seawater was at 10 degrees Celcius and 30‰ salinity Ovigerous Lepeophtheirus salmonis were collected from adult Atlantic salmon following harvest from a farm near Vancouver Island and transported in ice cold aerated seawater to Nanaimo. Dissected egg strings were incubated in filtered and ultraviolet irradiated seawater at 9.5 ± 1.0 C and 29.5 ± 0.5‰ salinity, with supplemental aeration, as described previously (Jones et al. 2006). Cultured lice were monitored by daily microscopic examination of triplicate samples and an inoculum containing a known number of copepodids was prepared when the ratio of copepodid to nauplius II stages was greatest.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from fin and kidney samples using Trizol, as per manufacturer’s instructions, and purified using RNeasy spin columns (QIAGEN), by manufacturer’s instructions with the on-column DNase I digestion. The RNA was quality checked by agarose gel electrophoresis and quantified by spectrometry (NanoDrop-1000).
|
| Label |
Cy3-CTP
|
| Label protocol |
Total RNA samples from Trial 3 were randomized and 200ng total RNA of each was reverse transcribed to cDNA and amplified to labelled-cRNA using Low Input Quick Amp labelling kits as per manufacturer’s instructions (Agilent). Reference channel Cy3-cRNA was constructed by combining equimolar cRNA proportions from each species/infection class conditions, and from each condition including at least one representative from each day (n = 19).
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized to randomized-order cGRASP 4x44k salmonid arrays (Agilent eArray AMADID: 025055) as per manufacturer’s instructions and slides were washed using stabilization solution to minimize ozone-related problems (Agilent; Sutherland et al., 2012). Slides were kept in the dark in a low ozone atmosphere until scanned.
|
| Scan protocol |
scanned on a ScanArray Express (Perkin Elmer) at constant PMT settings to produce saturated median values for ~1% of spots (anterior kidney samples: Cy3:80; Cy5:75 - skin samples: Cy3:80; Cy5:80).
|
| Description |
raw data filenames: 10214_cy5_70_scan2.txt, 10214_cy3_80_scan2.txt; block for sample in raw data file: Block3
Chm_AK_d6_con_rep2
|
| Data processing |
Images were quantified using Imagene (v8; BioDiscovery) and poor or empty spots were flagged. Per block, a background correction for all probes was performed by subtracting the block’s average signal median value for negative control spots from each probe. Sample files were then imported into GeneSpring (v11; Agilent). For each species and tissue experiment, (e.g. Atlantic salmon, head kidney) filters were applied to retain probes for which 65% or more of all samples within at least one condition had background-corrected raw expression values ≥500 in both channels and flag values for each channel as ‘present’. For statistics tests, a probe was deemed differentially expressed if it passed a Benjamini-Hochberg multiple test corrected p-value ≤ 0.05 and fold change ≥ 1.5. In experiments with a time component (e.g. Atlantic or pink salmon head kidney), a 2-way ANOVA was used to detect probes with a significant effect of infection and those with a time-infection interaction effect. Probes with a main effect but no interaction effect were filtered to retain only those that varied by 1.5 fold between control and experimental for at least one of the three time points. Probes with a significant time by infection interaction were filtered at each time point (FC >= 1.5).
|
| |
|
| Submission date |
Jun 26, 2013 |
| Last update date |
Feb 24, 2014 |
| 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) |
| GSE48337 |
Comparative transcriptomics of Atlantic Salmo salar, chum Onchorhynchus keta and pink salmon O. gorbuscha responding to salmon lice Lepeophtheirus salmonis infections |
|
