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| Status |
Public on Aug 31, 2022 |
| Title |
PKD_RBC#3 |
| Sample type |
RNA |
| |
|
| Source name |
PKD_RBC
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
tissue: red blood cells
age: juvenile fish of ~ 20 cm
disease state: (PKD) diseased individual
|
| Treatment protocol |
Upon arrival, fish were bled from caudal veins using heparinized syringes and needles, and euthanized with clove oil. Upon dissection, for three suspected cases of PKD out of 19 fish studied, we prepared imprints from their swollen kidneys and stained them according to the manufacturer’s instructions with the Kwik–Diff Kit (Richard Allen Scientific, San Diego, CA, USA), consisting of methanol fixation, followed by eosin and methylene blue staining. Light microscopy was used to identify T. bryosalmonae parasites. All fish in the cohort had kidney swelling indices between 3-4 according to the scale and parameters established by Clifton-Hadley et al. (1987) and variable signs of spleen swelling, fibrosis, and of gill and liver pallor.
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| Growth protocol |
Rainbow trout Oncorhynchus mykiss (mean body mass of 120 g) were reared in a commercial inland fish farm, in the South Bohemian region of the Czech Republic. Fish displaying gross signs of PKD such as pale gills, abdominal distension, and lethargy were transported alive to the Institute of Parasitology of the Biology Centre of the Czech Academy of Sciences (České Budějovice, Czech Republic). These fish were compared to an additional 15 naïve SPF fish (mean body mass of 30 g), reared in experimental recirculating systems of the Faculty of Fisheries and Protection of Waters, University of South Bohemia in the Czech Republic.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RBCs were separated from the whole blood of PKD and SPF rainbow trout. RBCs were washed three times with PBS, then aliquots of 25 × 10E6 cells were pelleted and stored in RNAlater (Sigma-Aldrich, Saint Louis, MO, USA) until further processing
|
| 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).
|
| 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 |
Mar 17, 2022 |
| Last update date |
Aug 31, 2022 |
| 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) |
| GSE198859 |
Analysis of red blood cells from rainbow trout Oncorhynchus mykiss infected by Tetracapsuloides bryosalmonae |
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