|
| Status |
Public on Jul 31, 2009 |
| Title |
D.rerio_adult_chronic (i1) vs control (c1) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
D.rerio_adult_control_c1
|
| Organism |
Danio rerio |
| Characteristics |
tissue: whole organism
age: adult
disease state: healthy
|
| Biomaterial provider |
MCB lab, IBL, Leiden
|
| Treatment protocol |
none
|
| Growth protocol |
Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Fish were snap frozen in liquid nitrogen, stored at -80°C, and homogenized in liquid nitrogen using a mortar and pestle. Portions of 50 -100 μg of powdered tissue were used for extraction of total RNA with 1 ml of TRIZOL® Reagent (Invitrogen) according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to clean up using RNeasy columns (Qiagen). The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies). The samples used had an average RIN value of 9.5 and a minimum RIN value of 8.9.
|
| Label |
Cy3
|
| Label protocol |
Amino Allyl modified aRNA was synthesized in one amplification round from 1 ug of total RNA using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion). Subsequently, 6 ug of Amino Allyl modified aRNA was used for coupling of monoreactive Cy3 dye (GE Healthcare) and column purified.
|
| |
|
| Channel 2 |
| Source name |
M.marinum_chronic_infected fish_i1
|
| Organism |
Danio rerio |
| Characteristics |
tissue: whole organism
age: adult
disease state: M. marinum infected fish showing overt signs of fish tuberculosis
|
| Biomaterial provider |
MCB lab, IBL, Leiden
|
| Treatment protocol |
Adult male zebrafish were infected by intraperitoneal inoculation with approximately 1x 10-3 M. marinum bacteria
|
| Growth protocol |
Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Fish were snap frozen in liquid nitrogen, stored at -80°C, and homogenized in liquid nitrogen using a mortar and pestle. Portions of 50 -100 μg of powdered tissue were used for extraction of total RNA with 1 ml of TRIZOL® Reagent (Invitrogen) according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to clean up using RNeasy columns (Qiagen). The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies). The samples used had an average RIN value of 9.5 and a minimum RIN value of 8.9.
|
| Label |
Cy5
|
| Label protocol |
Amino Allyl modified aRNA was synthesized in one amplification round from 1 ug of total RNA using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion). Subsequently, 6 ug of Amino Allyl modified aRNA was used for coupling of monoreactive Cy5 dye (GE Healthcare) and column purified.
|
| |
|
| |
| Hybridization protocol |
The dual colour hybridization of the microarray chips was performed at ServiceXS (ServiceXS, Leiden, The Netherlands) using the standard Agilent protocol.
|
| Scan protocol |
The arrays were scanned with DNA Microarray Scanner G2565CA from Agilent Technologies. The arrays were scanned twice with 10% PMT and 100% PMT laser power.
|
| Description |
D.rerio_adult_M.marinum_infected vs control replica_1
|
| Data processing |
Microarray data was processed from raw data image files with Feature Extraction Software 9.5.3 (Agilent Technologies). The XDR function was used to extend the dynamic range. Samples from control fish (c1-c4) were labelled with Cy3 dye and samples from infected fish (i1-i4) with Cy5 dye. After feature extraction, data were imported into Rosetta Resolver 7.1 (Rosetta Biosoftware, Seattle, Washington) and subjected to default ratio error modeling. Ratio results from four control fish versus four infected fish (c1 vs. i1, c2 vs. i2, c3 vs. i3, c4 vs. i4) were combined using the default ratio experiment builder.
|
| |
|
| Submission date |
Feb 10, 2009 |
| Last update date |
Jun 02, 2009 |
| Contact name |
Anna Magdalena Zakrzewska |
| E-mail(s) |
ania.zakrzewska@gmail.com
|
| Phone |
020-6923990
|
| Fax |
-
|
| URL |
http://-
|
| Organization name |
University of Leiden
|
| Department |
IBL
|
| Lab |
A. Meijer
|
| Street address |
Wassenaarseweg 64
|
| City |
Leiden |
| ZIP/Postal code |
2333 AL |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL7735 |
| Series (2) |
| GSE14782 |
Mycobacterium marinum chronic infection of adult zebrafish |
| GSE15328 |
Specificity of the zebrafish host transcriptome response to acute and chronic mycobacterial infection |
|
