|
| Status |
Public on Jun 04, 2017 |
| Title |
kidney_gradual stress_24°C_replicate2 |
| Sample type |
RNA |
| |
|
| Source name |
kidney gradual stress 24 C
|
| Organism |
Coregonus lavaretus |
| Characteristics |
tissue: kidney
gender: mixed
age: 349 days post hatch
|
| Treatment protocol |
Fish (30.3 ± 1.7 cm in length, and 300.6 ± 68.5 g in weight ) were randomly allocated to identical 300-L glass tanks (0.74 m length × 0.58 m width × 0.72 m height), receiving brackish water from the Darss-Zingst Bodden chain (2.5–6 practical salinity units) to the recirculating aquaculture system with an exchange rate of about 0.5 times per hour. Two tanks out of nine tanks were assigned as “temperature reference” tanks and kept at 18 °C for the entire duration of the experiments. Water temperature in two other tanks was elevated from 18 °C to 24 °C by 1 °C every two days, assigned as “gradual stress”. The remaining five tanks served to perform the experiment of “acute stress”. Water temperature in two of these tanks was kept constant at 18 °C for 18 d, but at day 19, whitefish were transferred from these tanks into two 24 °C-warm tanks, where fish was kept for 1 h until sampling. To account for handling procedures, fish were transferred in the same manner into the equally 18 °C-tempered tank (temperature reference). Fish were randomly sampled using hand nets. Anaesthetization of fish with was done using phenoxyethanol prior to tissue sampling.
|
| Growth protocol |
Fish were grown in water recirculation tanks at the Institute for Fisheries in Born, Germany.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Liver, spleen, kidney were sampled. 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 Fluidigm assays.
|
| Label |
Cy3
|
| Label protocol |
Seven individual RNA samples from the same tissue (liver, spleen, kidney) and treatment 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 7 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 |
Feb 27, 2017 |
| Last update date |
Jun 04, 2017 |
| 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) |
| GSE95422 |
Potential biomarker genes for acute and gradual temperature stress in maraena whitefish Coregonus lavaretus |
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