|
| Status |
Public on Oct 24, 2015 |
| Title |
Gills_HD_Born_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
gill filaments
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
tissue: gill filaments
gender: mixed
age: 10 months
strain: Born
density: high
|
| Treatment protocol |
Rainbow trout were randomly sampled using hand nets. Anaesthetization of fish with was done using phenoxyethanol prior to tissue sampling.
|
| Growth protocol |
Tank (0.74 m length × 0.58 m width × 0.72 m height) with a volume of ~310 L water received a continuous supply of running brackish water from the Baltic sea (2.5–6 practical salinity units) with a flow rate of 100–150 L/h. Water quality was monitored in all of the tanks throughout the trial. Light:dark period was 17.5:6.5. The water quality parameters were maintained as follows: temperature 16.4–19.8 °C, dissolved oxygen 9.8–10.5 mg/l, and pH 7.4–7.8. Trout were fed commercial dry pellets by automatic feeders located centrally above each tank distributing the food 12 h/d.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Entire liver; gill filaments; and head and trunk kidney were taken from ten Born and ten Troutlodge trout kept at MD, ED, and HD. RNeasy Mini Kit (Qiagen, Hilden, Germany) in complement with the RNase-free DNase Set (Qiagen) allowed us to obtain high-quality RNA suitable for microarray hybridizations and quantitative real-time PCR (qPCR) assays.
|
| Label |
Cy3
|
| Label protocol |
Five individual RNA samples from the same tissue (liver, gills, kidney), strain (Born or Troutlodge) and stocking density (MD, ED, HD) were pooled. 100 ng of each total RNA samples was amplified and labeled with the fluorescent dye cyanine 3 using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies). Yields of cRNA and the dye-incorporation rate were measured.
|
| |
|
| Hybridization protocol |
The hybridization procedure was performed using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Equal amount of fragmented Cy3-labeled cRNA (0.6 µg) in hybridization buffer was hybridized at 65 °C for 17 h to 8×60 K Whole Salmon Genome Oligo Microarrays (AMADID 049158; Agilent Technologies) using Agilent’s recommended hybridization chamber and oven. Following hybridization, the microarrays were washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 at 37 °C for 1 min.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using the Microarray Scanner System G2505C (Agilent Technologies).
|
| Description |
Pool of 5 individual RNAs
|
| Data processing |
The Agilent Feature Extraction Software 10.7.3.1 was used to read out and process the microarray image files using default settings. FES corrected the background based on a two-sided Student t-test.
|
| |
|
| Submission date |
Oct 23, 2015 |
| Last update date |
Oct 24, 2015 |
| Contact name |
Alexander Rebl |
| E-mail(s) |
rebl@fbn-dummerstorf.de
|
| Phone |
+493820868721
|
| Organization name |
Research Institute for Farm Animal Biology
|
| Department |
Institute of Genome Biology
|
| Lab |
Fish Genetics
|
| Street address |
Wilhelm-Stahl-Allee 2
|
| City |
Dummerstorf |
| ZIP/Postal code |
18196 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21057 |
| Series (1) |
| GSE74332 |
Potential biomarker genes for crowding stress in rainbow trout Oncorhynchus mykiss |
|
