|
| Status |
Public on Dec 02, 2015 |
| Title |
0°C kidney-TCO-replicate1 |
| Sample type |
RNA |
| |
|
| Source name |
0°C kidney-TCO
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
tissue: kidney
strain: TCO
gender: female
age: 8 months
|
| Treatment protocol |
Fish were put in basins containing either 5°C or 0°C cold water and incubated for 30 minutes. Fish were randomly sampled using hand nets. Anaesthetization of fish with was done using phenoxyethanol prior to tissue sampling.
|
| Growth protocol |
Fish were grown under standard aquauculture conditions in brackish water. When a temperature of 5°C was reached the treatment was performed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Liver, kidney and gill were taken from BORN and TCO. 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 TCO) and temperature (5°C, 0°C) 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 |
Dec 01, 2015 |
| Last update date |
Dec 02, 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) |
| GSE75563 |
Identification of genes involved in cold shock response in rainbow trout (Oncorhynchus mykiss) |
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