|
| Status |
Public on Jul 31, 2009 |
| Title |
D.rerio_adult_CR_vs_Mma20_6dpi_4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
D.rerio_adult_CR
|
| Organism |
Danio rerio |
| Characteristics |
reference: CR (common reference) is a mixture of all RNA samples from healthy and infected adult zebrafish used in this microarray study
|
| Biomaterial provider |
Astrid van der Sar, VUMC
|
| Treatment protocol |
This sample is a mixture of all RNA samples from early adult infection study (1 and 6 dpi infection with Mma20 and E11 strains and PBS-injected controls)
|
| 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 |
Adult fish were homogenized in liquid nitrogen using a mortar and pestle and 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. Embryos were directly homogenized in TRIZOL® Reagent. 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 purification using the RNeasy MinElute Cleanup kit (Qiagen) according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies). Samples used for microarray analysis had an average RIN value of 9 and a minimum RIN value of 8.
|
| Label |
Cy3
|
| Label protocol |
Amino Allyl modified aRNA was synthesized in one amplification round from 1 μg 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 |
D.rerio_adult_Mma20_6dpi
|
| Organism |
Danio rerio |
| Characteristics |
tissue: adult zebrafish
infection: 10000 bacteria of M.marinum Mma20 strain
time: 6 dpi (day post infection)
|
| Biomaterial provider |
Astrid van der Sar, VUMC
|
| Treatment protocol |
Zebrafish were inoculated intraperitoneally as previously described (Meijer et al., 2004) with approximately 10000 bacteria or with phosphate-buffered saline (PBS) as a control.
|
| Growth protocol |
Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org).
Infection experiments with adult fish were performed on young males selected from a wild type laboratory-breeding colony and acclimated to their new environment for one week in a quarantine area. These fish were kept at 28˚C on a 12:12 h light/dark rhythm throughout the experiment. Groups of 30 fish, infected with the same dose and strain of mycobacteria, were kept in small fish tanks (10 l) with their own separate filtering system (Eheim Ecco).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Adult fish were homogenized in liquid nitrogen using a mortar and pestle and 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. Embryos were directly homogenized in TRIZOL® Reagent. 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 purification using the RNeasy MinElute Cleanup kit (Qiagen) according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies). Samples used for microarray analysis had an average RIN value of 9 and a minimum RIN value of 8.
|
| Label |
Cy5
|
| Label protocol |
Amino Allyl modified aRNA was synthesized in one amplification round from 1 μg 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) according to Agilent protocol G4140-90050 v.5.7 (www.Agilent.com) for Two-Color Microarray-Based Gene Expression Analysis.
|
| Scan protocol |
Microarray data were processed from raw data image files with Feature Extraction Software 9.5.3 (Agilent Technologies).
|
| Description |
D.rerio_adult_CR_vs_Mma20_6dpi_4
|
| Data processing |
Processed data were subsequently imported into Rosetta Resolver 7.1 (Rosetta Biosoftware, Seattle, Washington) and subjected to default ratio error modelling. Data were analyzed at the level of UniGene clusters (UniGene build #105). The significance cut-off for ratios of infected versus control was set at P ≤ 10-4. For adult infection studies, where triplicate individuals are tested, an absolute fold change cut-off (≥ 1.5) was applied additionally.
|
| |
|
| Submission date |
Mar 17, 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 (1) |
| GSE15328 |
Specificity of the zebrafish host transcriptome response to acute and chronic mycobacterial infection |
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