|
| Status |
Public on Apr 23, 2015 |
| Title |
C1_F2 |
| Sample type |
RNA |
| |
|
| Source name |
4 months old zebrafish liver
|
| Organism |
Danio rerio |
| Characteristics |
gender: female
agent: 5-FU
dose: 0.01 µg/L
age: 4 months old
tissue: liver
|
| Treatment protocol |
5-fuorouracil (5-FU) (CAS 51-21-8) was purchased from Sigma-Aldrich. Stock solution (100 mg/L) was prepared in dimethyl-sulfoxide (DMSO) and used for toxicity evaluation after dilution in clean system water (see parameters above). The nominal exposure concentrations were 0.01 and 1 a100 µg/L of 5-FU. The control group of fish was kept in the system water supplemented with 1μl/L of DMSO (vehicle control). All treatments including controls were conducted in duplicates (A and B). The experiment was performed under the semistatic conditions with a complete renewal of exposure medium every three days. Chemical analyses of treatment medium samples were performed immediately after the medium exchange, at the end of the second day (48 h) and the end of the third day (72 h). At the age of 4 month part of F1 fish was sampled for gene expression analyses.
|
| Growth protocol |
AB line of zebrafish (Danio rerio) that was used in the experiments, originated from the Department of Aquaculture of Szent István University. Prior to the experiment, the parent generation was maintained in a recirculation system allowing continuous flow of water (ZebTec, Tecniplast Inc.) in a light regime of 14 h light/10 h dark. Parameters of the system water were as follows: temperature 25±0.5 °C, pH 7.4±0.2, conductivity: 525±50 µS. During the experimental period fish were maintained at 24-26 °C in a light regime of 14 h light/10 h dark.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA was isolated by Direct-zol RNA Miniprep (ZymoResearch) from liver from 10 individuals per exposure point from each of the two replicates. After RNA quality and quantity evaluation (Agilent Bioanalyzer, RNA 6000 Nano Kit, Eukaryote Assay), RNA from 3 individuals from same treatment, replicate and gender was pooled, DNase treated (Invitrogen DNaseI; 0.2 U/µg RNA) in 45 μl reactions and subsequently purified using RNeasy Mini Elute kit (Qiagen). Purified RNA was checked for integrity and quantity by Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
The total RNA was labelled with Cy-3 using Low Input Quick-Amp Labeling Kit One-Color (Agilent technologies) following manufacturers protocol
|
| |
|
| Hybridization protocol |
Hybridizations and washes were performed using Gene Expression Hybridization Kit (Agilent Technologies) following manufactures protocol
|
| Scan protocol |
Hybridized slides were scanned on Agilent DNA Microarray Scanner and signals were extracted and quality control was performed using Feature Extraction 10.7.3.1 Software (Agilent Technologies).
|
| Description |
pool of 3 individuals
|
| Data processing |
Microarray data analysis was performed in R environment (version 2.15.1) using Bioconductor’s libraries limma and vsn.
|
| |
|
| Submission date |
Nov 06, 2014 |
| Last update date |
Apr 24, 2015 |
| Contact name |
Spela Baebler |
| E-mail(s) |
spela.baebler@nib.si
|
| Organization name |
National Institute of Biology
|
| Department |
Department of Biotechnology and Systems Biology
|
| Street address |
Vecna pot 111
|
| City |
Ljubljana |
| ZIP/Postal code |
1000 |
| Country |
Slovenia |
| |
|
| Platform ID |
GPL14664 |
| Series (1) |
| GSE63039 |
Zebrafish liver transcriptome in response to chronic treatment with environmentally relevant 5-FU |
|
