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| Status |
Public on Sep 01, 2021 |
| Title |
3h control_#3 |
| Sample type |
RNA |
| |
|
| Source name |
control fish (w/o stress)
|
| Organism |
Coregonus maraena |
| Characteristics |
tissue: head kidney
gender: unknown
age: juvenile fish of ~ 20 cm
|
| Treatment protocol |
The fish (n = 48 in total) used in the experiments were juvenile with a starting size of about 20 cm. They were allowed to acclimatize to the recirculation system and the experimental tanks for at least 3 weeks. Experiments were always carried out in the morning (between 8 a.m. and 11 a.m.). To avoid tank effects, fish were placed in pairs consisting of one control fish and one test fish in identical dark-polyethene tanks (2 × 8 pairs; n = 32) during the acute stress experiments (3 h and 24 h). At the start of the experiment and before the handling procedure, one fish per pair was euthanized. This fish was designated the control fish (0 h). Following the sampling of the control fish, the stress protocol was applied for one minute to the remaining fish, designated the test fish. The stress protocol consisted of hunting and catching fish inside the water with nets, intermittently lifting the fish out of the tanks and finally transferring fish into secondary tanks, where pairs of test fish were left undisturbed for either 3 h or 24 h. After this period, test fish were euthanized and tissues were sampled as described below (see 2.3.). For the chronic handling stress experiment, control fish (n = 8) and test fish (n = 8) were kept in separate tanks for the duration of the experiment. Test fish were hunted and netted in the water for one minute once a day for ten days, while the controls were left undisturbed. The net lifting and tank-transferring steps performed in the acute-stress procedure were skipped in the chronic stress experiment to avoid skin injuries. To minimize habituation, handling was always performed at a random time within the 12 h ‘daylight’ period. Fish were euthanized 10 days after the onset of the chronic-stress procedures, 24 h after the last handling procedure.
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| Growth protocol |
Fish were grown in water recirculation tanks of the Fisheries of the State Research Center for Agriculture and Fishery Mecklenburg-Western Pomerania (Born, Germany) and BiMES - Binnenfischerei GmbH (Friedrichsruhe, Germany). Fish were reared in fresh-water recirculation systems with a stocking density of 10 kg/m³ at 18 °C and a 12:12-h day-night cycle. Water quality was maintained by automated purification and disinfection (bio-filter and UV light).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Fish was dissected and a portion of the head kidney was snap-frozen in liquid nitrogen. 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.
|
| Label |
Cy3
|
| Label protocol |
50 ng of RNA samples was amplified and labeled with the fluorescent dye cyanine 3 using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) following the Low Input Quick Amp protocol. Yields of cRNA and the dye-incorporation rate were measured.
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| |
|
| Hybridization protocol |
The hybridization procedure was performed using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Equal amount of fragmented Cy3-labeled cRNA in hybridization buffer was hybridized to 8×60 K Whole Salmon Genome Oligo Microarrays (ID 020938, 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 |
control fish (w/o stress)
|
| Data processing |
The Agilent Feature Extraction Software 12.1.1.1 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.
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| |
|
| Submission date |
Aug 31, 2021 |
| Last update date |
Sep 01, 2021 |
| 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) |
| GSE183125 |
Microarray-predicted marker genes indicating handling stress in maraena whitefish (Coregonus maraena) |
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