|
| Status |
Public on Oct 01, 2011 |
| Title |
Embryo E. coli immersed rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
D.rerio_embryo_CR
|
| Organism |
Danio rerio |
| Characteristics |
comment: 24 hpf embryos were dechorionated with pronase (2 mg/ml) and at 25 hpf immersed for 5 hours in pure eggwater (60 µg/ml Instant Ocean salts) or eggwater containing 1E8 CFU/ml of E. tarda or E. coli or 1E9 CFU/ml of P. aeruginosa PAO1 or PA14. Embryos were then quenched in liquid nitrogen and stored at -80˚C prior to RNA extraction. This sample is a mixture of all RNA samples used in this study.
sample type: pooled common reference
|
| Treatment protocol |
Zebrafish embryos used for the bacterial immersion assay were dechorionated with pronase (2 mg/ml) at 24 hpf. At 25 hpf they were immersed in bacterial suspensions of E. tarda (1E8 CFU/ml), E. coli (1E8 CFU/ml), Pseudomonas aeruginosa PAO1 (1E9 CFU/ml) or P. aeruginosa PA14 (1E9 CFU/ml) for 5 h after which the embryos were quenched in liquid nitrogen.
|
| Growth protocol |
Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). Embryos were grown at 28,5°C in egg water (60µg/ml Instant Ocean sea salts).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent (Invitrogen) and subsequently total RNA was extracted 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 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).
|
| 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 or Cy5 dye (GE Healthcare) and column purified.
|
| |
|
| Channel 2 |
| Source name |
D.rerio_embryo_e.coli_5hpi_2
|
| Organism |
Danio rerio |
| Characteristics |
tissue: embryo
time point: 30 hpf
infection: 1E8 CFU/ml of E. coli
tissue: embryo
|
| Treatment protocol |
Zebrafish embryos used for the bacterial immersion assay were dechorionated with pronase (2 mg/ml) at 24 hpf. At 25 hpf they were immersed in bacterial suspensions of E. tarda (1E8 CFU/ml), E. coli (1E8 CFU/ml), Pseudomonas aeruginosa PAO1 (1E9 CFU/ml) or P. aeruginosa PA14 (1E9 CFU/ml) for 5 h after which the embryos were quenched in liquid nitrogen.
|
| Growth protocol |
Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). Embryos were grown at 28,5°C in egg water (60µg/ml Instant Ocean sea salts).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent (Invitrogen) and subsequently total RNA was extracted 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 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).
|
| 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 Cy3 or 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 |
The CR (common reference) was composed by combining RNA from each sample of the immersion experiment
Biological replicate 2 of 3: Zebrafish embryos infected with E. coli
|
| Data processing |
Microarray data was processed from raw data image files with Feature Extraction Software 9.5.3.1 (Agilent Technologies). The XDR function was used to extend the dynamic range. Processed data were subsequently imported into Rosetta Resolver 7.0 (Rosetta Biosoftware, Seattle, Washington) and subjected to default ratio error modelling.
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| |
|
| Submission date |
Apr 08, 2011 |
| Last update date |
Oct 01, 2011 |
| Contact name |
Oliver Stockhammer |
| E-mail(s) |
o.w.stockhammer@amc.uva.nl
|
| Organization name |
AMC
|
| Department |
Medical Microbiology
|
| Street address |
Meibergdreef 15
|
| City |
Amsterdam |
| State/province |
NH |
| ZIP/Postal code |
1105 AZ |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL13389 |
| Series (2) |
| GSE28481 |
Transcriptome analysis of the zebrafish embryonic host response to Edwardsiella tarda infection using a static immersion systems [experiment A] |
| GSE28486 |
Transcriptome analysis of the zebrafish embryonic host response to Edwardsiella tarda infection |
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