|
| Status |
Public on Apr 04, 2019 |
| Title |
spleen_temperature stress_27°C_tech-replicate1 |
| Sample type |
RNA |
| |
|
| Source name |
spleen_temperature stress_27°C_tech-replicate1
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
tissue: spleen
gender: mixed
age: ~10 months
|
| Treatment protocol |
Fish (26.2 ± 1.3 cm in length, and 230.2 ± 35.9 g in weight ) were randomly allocated to identical 309-L glass tanks (0.74 m length × 0.58 m width × 0.72 m height). The experimental procedures are illustrated in Fig. 1(Overall design diagram.pdf). The stocking density in four of the six experimental tanks (A1, A2, B1, B2) has been set to 30 kg/m3 (corresponding to 38 or 39 trout weighing 9.5 kg), while the stocking density in the other two of the six tanks (C1, C2) was 100 kg/m3 (corresponding to 96 or 119 trout weighing 35 kg). Two of the tanks with 30 kg of trout per cubic metre (A1, A2) served as control tanks and were kept at a constant temperature of 16 °C for the entire duration of the experiment. The water temperature in the other four tanks B1, B2 and C1, C2 was gradually elevated from 16 °C to 26 °C by 2 °C every day for a period of 6 days. On day 7, the temperature of 27 °C was kept for 24 h. Further husbandry conditions are detailed in reference (Rebl et al., 2017: Microarray-predicted marker genes and molecular pathways indicating crowding stress in rainbow trout (Oncorhynchus mykiss). Aquaculture 473, 355–365). The water temperature and other relevant parameters were constantly recorded. Trout were fed ad libitum commercial dry pellets by automatic feeders. Throughout the experiment, no technical problems were registered.
|
| Growth protocol |
Fish were grown in water recirculation tanks at the Institute for Fisheries in Born, Germany.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Spleen was 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 spleen were pooled according to the different treatment groups. 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. All hybridisations include thus two biological and two technical replicates. The technical replicates in our study are exactly the same probes prepared for paired bio-replicate and were applied to an independent array.
|
| |
|
| 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 |
Apr 03, 2019 |
| Last update date |
Apr 04, 2019 |
| 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) |
| GSE129271 |
Microarray-predicted marker genes and molecular pathways indicating combined crowding and thermal stress in rainbow trout (Oncorhynchus mykiss) |
|
