|
| Status |
Public on Feb 18, 2022 |
| Title |
Vibrio vulnificus WT strain infection_WBC_0hpi_rep2 [CKS_47] |
| Sample type |
RNA |
| |
|
| Source name |
Isolated white blood cells,V.vulnificus WT strain infected, 0hpi, rep2
|
| Organism |
Anguilla anguilla |
| Characteristics |
tissue: Whole blood
cell type: Isolated white blood cells
treatment: V.vulnificus WT strain infected, 0hpi
|
| Treatment protocol |
For transcriptomic experiments, eels of 100 g were distributed into two groups, the tested (n= 24 individuals) and the control group (n= 6 individuals). Individuals were then immersed either in an infective bath containing 2x106 CFU of the strain R99 (the previously estimated LD50) (tested group) or in sterilized SW (control group). After 1 h of immersion, fish were transferred separately into new tanks and kept under constant conditions until sampling.
|
| Growth protocol |
Adult European eels (Anguilla anguilla) of around 100 g of body weight were purchased from a local eel-farm that does not vaccinate against V. vulnificus. Eel maintenance and all the experiments described above were performed at 28°C in 180-liter tanks containing either 60 (infection experiments) or 120 L (the rest of experiments and animal maintenance) of saline water (SW, 1.5% NaCl, pH 7) with a system of aeration and filtration in the facilities of the Central Service for Experimental Research (SCSIE) of the University of Valencia (Spain).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (from eels B, RBCs, and WBCs obtained at 0, 3, and 12 hpi) was extracted with NucleoZOL (Macherey-Nagel) following the manufacturer’s instructions. Possible contaminating DNA was eliminated using TURBOTM DNase (Ambion) and then RNA was cleaned with RNA Cleanup and Concentration Micro Kit RNA (Thermo Scientific) according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
RNA samples were labelled with a single dye, Cy3. Denatured samples of RNA were reversed transcribed and indirectly labelled with Cy3. RNA labelling were performed according to agilent Quick Amp Labelling kit instructions.
|
| |
|
| Hybridization protocol |
1.65 μg of cDNA-labbled of each sample was used for the hybridization to custom european eel array during 17h at 65ºC following the instructions of Agilent’s GE Hybridization Kit.
|
| Scan protocol |
Microarrays slides were scanned with Agilent Technologies Scanner model G2505B. Spot intensities and other quality control features were extracted with Agilent’s Feature Extraction software version 10.4.0.0. Txt files generated were imported into the Gene GeneSpring software GX 12.5.
|
| Description |
Gene expression after 0hr in V.vulnificus WT strain (strain R99) infected isolated white blood cells.
|
| Data processing |
Percentile shift normalization was made to adjust all spot intensities in an array. This normalization takes each column in an experiment independently, and computes the median expression values for this array, across all spots. It then subtracts this value from the expression value of each entity (Bolstad et al, 2003). After Percentile normalization, all data were filtered by comparison of the standard deviation expression among groups (filter by expression). The entities that had values lesser or greater than the standard deviation value were retained. This filter procedure allowed selected genes that have outlier samples, and filter out genes that have a low variation in expression values across all samples.
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| |
|
| Submission date |
Feb 17, 2022 |
| Last update date |
Feb 18, 2022 |
| Contact name |
Carmen Amaro |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Valencia
|
| Department |
Microbiology and Ecology
|
| Street address |
Dr. Moliner, 50
|
| City |
Burjassot |
| State/province |
Valencia |
| ZIP/Postal code |
46100 |
| Country |
Spain |
| |
|
| Platform ID |
GPL16775 |
| Series (1) |
| GSE196944 |
A transcriptomic study reveals that fish vibriosis due to the zoonotic pathogen Vibrio vulnificus is an acute inflammatory disease. |
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