|
| Status |
Public on Sep 22, 2009 |
| Title |
Brain_animal-4453_line-61 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Brain_animal-4453_low_feather_pecking_selection_line
|
| Organism |
Gallus gallus |
| Characteristics |
strain: White Leghorn Low Feather Pecking line (Gallus gallus domesticus); This is a line which is selected for a decrease in feather pecking behaviour for 8 generations
gender: female
age: 34 weeks
tissue: whole brain
severe feather pecking: 2
aggressive feather pecking: 0
gentle feather pecking: 2
receiving severe pecks: 0
receiving aggressive pecks: 0
receiving gentle pecks: 0
cage: 6
weight_g: 1205.1
|
| Biomaterial provider |
The sample was obtained by A.J. Buitenhuis from Aarhus University, Denmark.
|
| Treatment protocol |
From 1-day-old until 18 weeks of age: group housing. 18 weeks - 33 weeks of age: individual housing to collect the eggs. At 33 weeks of age the chickens were divided into groups of 20 hens for each line, separately. The birds had a week to get used to the new environment and group composition. At 34 weeks of age the chickens were subject to a social feather pecking test of 3h between 14:00h to 17:00h. This test is used in the selection procedure of the selection lines and is described in detail by Kjaer et al. [Kjaer, J.B., Sørensen, P, and G. Su. 2001. Divergent selection on feather pecking behaviour in laying hens (Gallus gallus domesticus). Appl. Anim. Behav. Sci. 71: 229-239]. The next day between 8:00h and 12:00h the bird was decapitated and the whole brain was taken and frozen directly in liquid nitrogen and stored at - 80 C until further use.
|
| Growth protocol |
The birds were reared in floor pens covered with a 5-cm thick layer of wood shavings. The temperature was 34 C at 1-day-old and gradually reduced to 20 C at 8 weeks of age. This temperature was kept throughout the rest of the experiment. The light regime was 12L:12D from 0 to 14 weeks and then 1 hour light per week was added until the light regime was 16L:8D at 18 weeks of age. Thereafter the light regime was constant until the bird was used in the experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The whole brain was homogenized in liquid nitrogen using a TissueLyser (Qiagen/Retsch) fitted with 50 mL stainless steel grinding jars and 20 mm grinding balls. Total-RNA was purified and DNase treated using NucleoSpin RNA L (Macherey-Nagel) following the enclosed protocol.
|
| Label |
Alexa-647
|
| Label protocol |
Ten µg Total-RNA from each individual sample was labeled with Alexa-647 using the SuperScript Direct cDNA labeling System (Invitrogen). The labelled cDNA was purified using the NucleoSpin 96 Extract II PCR Clean-up kit (Macherey-Nagel).
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| |
|
| Channel 2 |
| Source name |
Brain_reference
|
| Organism |
Gallus gallus |
| Characteristics |
strain: White Leghorn F1 animal from a cross between high feather pecking line x low feather pecking line (Gallus gallus domesticus)
gender: female
age: 34 weeks
tissue: whole brain.
|
| Biomaterial provider |
The sample was obtained by A.J. Buitenhuis from Aarhus Unversity, Denmark.
|
| Treatment protocol |
From 1-day-old until 18 weeks of age: group housing. 18 weeks - 34 weeks of age: individual housing to collect the eggs. At 34 weeks of age: the bird was taken out of his cage and between 8:00h and 12:00h the bird was decapitated and the whole brain was taken and frozen directly in liquid nitrogen and stored at - 80 C until further use.
|
| Growth protocol |
The birds were reared in floor pens covered with a 5-cm thick layer of wood shavings. The temperature was 34 C at 1-day-old and gradually reduced to 20 C at 8 weeks of age. This temperature was kept throughout the rest of the experiment. The light regime was 12L:12D from 0 to 14 weeks and then 1 hour light per week was added until the light regime was 16L:8D at 18 weeks of age. Thereafter the light regime was constant until the bird was used in the experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The whole brain was homogenized in liquid nitrogen using a TissueLyser (Qiagen/Retsch) fitted with 50 mL stainless steel grinding jars and 20 mm grinding balls. Total-RNA was purified and DNase treated using NucleoSpin RNA L (Macherey-Nagel) following the enclosed protocol.
|
| Label |
Alexa-555
|
| Label protocol |
Ten µg Total-RNA from the reference sample was labelled with Alexa-555 using the SuperScript Direct cDNA labeling System (Invitrogen). The labelled cDNA was purified using the NucleoSpin 96 Extract II PCR Clean-up kit (Macherey-Nagel). The labelled reference samples were mixed and divided into aliquots before combining it with the labelled sample.
|
| |
|
| |
| Hybridization protocol |
The slides were hybridized in a Discovery XT hybridization station (Ventana Discovery Systems, Tucson, AZ, USA). Transfer Chip Prep-2 from 4 ºC to room temperature 1 hour before use. Prepare ChipSpread by mixing equal volumes of ChipSpread A (20 mg/mL BSA, 4x SSC, 0.5 mg/mL sodium azide) and B (formamide; 2 mg/mL SDS) and incubate at room temperature for 1 hour before use. A total of 2.5 mL is needed per slide. Print labels, trim them and place them on the slides. Mix the Chip Map reagents (Chip Prep-1, -2 and - 3) by inversion, remove the cap and place the reagents in the Discovery. Place the slides in the machine and initiate the run. Cover slide with 2.5 mL ChipSpread when the message appears (after few minutes). The machine now runs for app. 1.5 hours to pre-hybridize the slides. Heat a waterbath to 90°C or use a PCR machine. Mix the Chiphybe, add 200 µL to the sample (<20 µL) and mix carefully. Heat the sample mixture at 90°C for 3 minutes and mix carefully by pipetting. Press "button" on the machine which then prepares the slides for hybridization. When the message appears apply 100 µL Chiphybe onto each slide. Then apply the samples (200 µL) onto each slide, press "button" and the machine hybridizes at 45 ºC for 6 hours. Wipe oil from backside of slides using a clean-room napkin and place slides in the slide-holder from the High Throughput Wash Station (Telechem, cat.no. HTW) placed in a mTub filled with RiboWash. If processing more than 20 slides, place equal number of slides in two slide-holders and continue in parallel. Dip the slides 20 times in RiboWash. Transfer the slide-holder to a HTW filled with RiboWash and wash for 2 min with magnetic stirring at 700 rpm. Refill the HTW with RiboWash and repeat the wash. Dip the slide-holder in 2x SSC filled in a mTub (200 mL 20x SSC, Elga H2O + 1800 mL water). Transfer the slide-holder to a HTW filled with 2x SSC and wash for 2 min with magnetic stirring at 700 rpm. Refill the HTW with 2x SSC and repeat the wash. Dip the slide-holder 10 times in 0.1x SSC filled in a mTub (5 mL 20x SSC, Elga H2O + 995 mL water) and leave the holder submerged in 0.1x SSC. Transfer the slides to a mBox slide holder placed in a mTub filled with Elga H2O. Dry arrays by centrifugation (at 300 x g for 4 min placed in a mBox).
|
| Scan protocol |
Scanner: ScanArray Express HT system (version 3.0, Perkin Elmer), 5 µm resolution, 100 % laser power and PMT adjusted individually for each channel. Image analysis software: GenePix Pro (version 6.0.1.27, Molecular Devices) using FeatureType = Irregular Filled and MorphologicalClosingFollowedByOpening as background measurement.
|
| Description |
Expression profiling of 179 chicken brains sampled from three lines - 60 chickens from a high feather pecking line (HP), 60 from a low feather pecking line (LP) and 59 from a random bred control line (C). Expression profiling was conducted by hybridising each sample together with a common reference, consisting of brain RNA from an unrelated F1 animal
|
| Data processing |
Statistical analysis was carried out in the R computing environment (version 2.5.0 for Windows) using the package Linear Models for Microarray Analysis (Limma, version 2.10.0) which is part of the Bioconductor project. The log2-transformed ratios of Alexa-647 to Alexa-555 (median intensities, not background corrected) were normalized within-slide using loess with default parameters as implemented in Limma. Further analysis: Single gene analysis: Limma version 2.10.0; Gene set enrichment analysis : Limma version 2.10.0 (GO/KEGG) / topGO version 1.2.1; 2 D clustering: heatmap.2 option in gplots version 2.3.2; co-expression: coXpress version 1.3.
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| |
|
| Submission date |
Feb 05, 2008 |
| Last update date |
Mar 09, 2010 |
| Contact name |
Jakob Hedegaard |
| E-mail(s) |
Jakob.Hedegaard@ki.au.dk
|
| Phone |
(+45)89991363
|
| Organization name |
Aarhus University, Faculty of Agricultural Sciences
|
| Department |
Department of Genetics and Biotechnology
|
| Lab |
Molecular Genetics and System Biology
|
| Street address |
PO-box 50
|
| City |
Tjele |
| ZIP/Postal code |
DK-8830 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL5480 |
| Series (1) |
| GSE10380 |
Analysis of severe feather pecking behavior in a high feather pecking selection line |
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