|
| Status |
Public on Jun 17, 2012 |
| Title |
High estrogen, fish number 325 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Liver of infected fish, high dose of E2
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
treated with: high dose (20 mg/kg feed) of E2 (17β-estradiol)
infected with: 10^6 cfu of Yersinia ruckeri
tissue: liver
|
| Treatment protocol |
Feed was spiked with E2 using alcohol evaporation method. For infection, the fish (n = 75 / treatment group) were transferred and kept in aerated 30 liter tanks/replicate filled with 5 liter of normal tap water with bacteria for 1 hour and then re-transferred to their original tanks. Three concentrations were applied: 10exp2 (low), 10exp4 (intermediate) and 10exp6 (high) colony forming units. Liver from fish exposed to high concentration was used for microarray analyses. Samples were collected after 10 days.
|
| Growth protocol |
Juvenile rainbow trout (1.15 g) was kept in aerated glass tanks with a flow-through of 1 liter/sec of normal tap water at temperature 13.8C. Fish was fed with dry feeds at 3% body weight / day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified using Trizol Plus/PureLink RNA Mini Kit (Invitrogen, Basel, Switzerland)
|
| Label |
Cy5
|
| Label protocol |
10 μg RNA in each were labeled with Cy3-dUTP (pooled sample of untreated fish used as reference) and Cy5-dUTP (fish from the exposed groups) (Amersham Biosciences, UK); labels were introduced during cDNA synthesis using SuperScript III RT (Invitrogen, Basel, Switzerland). The cDNA synthesis was performed at 46°C for 3 hours in 25 μl reaction volumes. RNA was degraded by adding 2.5M NaOH at 37°C for 15 min and alkaline was neutralized with 2M HEPES buffer.
|
| |
|
| Channel 2 |
| Source name |
Liver of control fish (no pathogen, no E2), pooled sample
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
sample type: pooled control smaple
tissue: liver
|
| Treatment protocol |
Feed was spiked with E2 using alcohol evaporation method. For infection, the fish (n = 75 / treatment group) were transferred and kept in aerated 30 liter tanks/replicate filled with 5 liter of normal tap water with bacteria for 1 hour and then re-transferred to their original tanks. Three concentrations were applied: 10exp2 (low), 10exp4 (intermediate) and 10exp6 (high) colony forming units. Liver from fish exposed to high concentration was used for microarray analyses. Samples were collected after 10 days.
|
| Growth protocol |
Juvenile rainbow trout (1.15 g) was kept in aerated glass tanks with a flow-through of 1 liter/sec of normal tap water at temperature 13.8C. Fish was fed with dry feeds at 3% body weight / day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified using Trizol Plus/PureLink RNA Mini Kit (Invitrogen, Basel, Switzerland)
|
| Label |
Cy3
|
| Label protocol |
10 μg RNA in each were labeled with Cy3-dUTP (pooled sample of untreated fish used as reference) and Cy5-dUTP (fish from the exposed groups) (Amersham Biosciences, UK); labels were introduced during cDNA synthesis using SuperScript III RT (Invitrogen, Basel, Switzerland). The cDNA synthesis was performed at 46°C for 3 hours in 25 μl reaction volumes. RNA was degraded by adding 2.5M NaOH at 37°C for 15 min and alkaline was neutralized with 2M HEPES buffer.
|
| |
|
| |
| Hybridization protocol |
Targets were combined and purified using Microcon YM-30 filter (Millipore, Billerica, USA). The microarray slides were pretreated with blocking solution (1% BSA fraction V, 20x SSC and 5% SDS) at 50°C for 30 min, then washed with 2x SSC (3 min) and 0.2xSSC (3 min). For hybridization, Lifter Slips (Erie Scientific, Portsmouth, USA) were placed on the slides. Labels were adjusted to 80 µl volumes and contained 1.3x Denhardt’s, 3x SSC, 0.3% SDS, 0.67 μg/μl polyadenylate and 1.4 μg/μl yeast tRNA. Hybridization was carried out overnight at 60°C in a water bath; the ArrayIt® Hybridization Chamber was used. Next, slides were washed in 0.5x SSC/0.1% SDS (15 min), 0.5x SSC/0.01% SDS (15 min) and twice in 0.06x SSC (3 min each) at room temperature in dim lighting with gentle agitation. Slides were dried using ArrayIt® Microarray High-Speed Centrifuge.
|
| Scan protocol |
Scanning was performed on a GenePix Personal 4100A microarray scanner (Molecular Devices, California, USA)
|
| Description |
H325
|
| Data processing |
The raw data file provided only contains the raw data spots that passed quality control. Images were processed using the standard procedures of GenePix Pro 6.0. After removal of flagged spots lowess normalization was performed
|
| |
|
| Submission date |
Jun 15, 2012 |
| Last update date |
Jun 17, 2012 |
| Contact name |
Aleksei Krasnov |
| E-mail(s) |
aleksei.krasnov@nofima.no
|
| Phone |
+47 97602165
|
| Organization name |
Nofima AS
|
| Department |
Fish Health
|
| Street address |
Osloveien 1
|
| City |
Aas |
| ZIP/Postal code |
1430 |
| Country |
Norway |
| |
|
| Platform ID |
GPL6154 |
| Series (1) |
| GSE38763 |
Estrogen modulates hepatic gene expression and survival of rainbow trout infected with pathogenic bacteria Yersinia ruckeri. |
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