|
| Status |
Public on Apr 10, 2014 |
| Title |
Isolation, rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
socially isolated fish_brain
|
| Organism |
Danio rerio |
| Characteristics |
strain: AB
genotype/variation: wild type
phenotype regarding social experience: socially isolated fish (I)
tissue: whole brain
|
| Treatment protocol |
In our study we used a modified version of an already established and reliable behavioural paradigm (see Oliveira et al., 2011) to study the influence of an acute agonistic interaction on the neurogenomic state of the adult zebrafish brain. In the present study, fish were isolated five days prior to the interaction. To control for the outcome perception experienced by fighting dyads, two other groups were used, an isolation group and a mirror directed aggression group. Mirror image stimulation has been known to elicit aggressive responses in fish, which do not recognize their own image and behave as in the presence of an intruder.
|
| Growth protocol |
All subjects used in this experiment were wild-type (AB) zebrafish (Danio rerio) acquired from Zebrafish International Resource Center (ZIRC). Prior to the experiment, animals were quarantined in 40L tanks (50x30x35cm) in a balanced sex ratio (1:1) for a period of two weeks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
biotin
|
| Label protocol |
RNA was processed for use on Affymetrix (Santa Clara, CA, USA) GeneChip Zebrafish Genome Arrays, according to the manufacturer’s GeneChip 3’ IVT Express kit user manual. Briefly, 100 ng of total RNA containing spiked in Poly-A RNA controls was used in a reverse transcription reaction (GeneChip 3’ IVT Express Kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in a 16h in vitro transcription (IVT) reaction to generate aRNA (GeneChip 3’ IVT Express Kit; Affymetrix). Size distribution of the aRNA and fragmented aRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
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| |
|
| Hybridization protocol |
15 µg of fragmented aRNA was used in a 250-µl hybridization cocktail containing added hybridization controls. 200 µl of mixture was hybridized on arrays for 16 h at 45°C. Standard post hybridization wash and double-stain protocols (FS450_0004; GeneChip HWS kit, Affymetrix) were used on an Affymetrix GeneChip Fluidics Station 450.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G. Scanned arrays were analyzed first with Affymetrix Expression Console software to obtain Absent/Present calls and to assure that all quality parameters were in the recommended range.
|
| Description |
I3
|
| Data processing |
Subsequent analysis was carried out with Partek Genomics Suite 6.6. A contrast analysis between the reference group (Isolation) and the target groups (winner, loser, and mirror) were performed using a two-way ANOVA for clustering the groups. For all the gene lists, genes were considered differentially expressed under a false discovery rate of 10% at a minimal fold change of 1.1.
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| |
|
| Submission date |
Apr 07, 2014 |
| Last update date |
Apr 10, 2014 |
| Contact name |
José Miguel Simões |
| Organization name |
ISPA - Instituto Universitário
|
| Department |
Eco-Ethology Research Unit
|
| Lab |
Integrative Behavioural Biology Group
|
| Street address |
Rua Jardim do Tabaco 34
|
| City |
Lisboa |
| ZIP/Postal code |
1140-041 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL1319 |
| Series (1) |
| GSE56549 |
Perception of fight outcome is needed to activate socially driven changes in brain transcriptome |
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