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| Status |
Public on Mar 02, 2009 |
| Title |
D.rerio_embryo_CR vs PBS 24hpi B |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
D.rerio_embryo_CR
|
| Organism |
Danio rerio |
| Characteristics |
CR (common reference) is a mixture of all RNA samples from this infection study.
|
| Biomaterial provider |
Oliver Stockhammer, IBL, Leiden University
|
| Treatment protocol |
The zebrafish embryos injected at 27 hours post fertilization with either PBS, Salmonella typhimurium WT or Salmonella typhimurium Ra mutant were collected 2, 5, 8 or 24 hours post injection and immediately 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 infection study
|
| 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 -30 °C in egg water (60µg/ml Instant Ocean sea salts) For the duration of PBS or bacterial injections embryos were kept under anaesthesia in egg water containing 0.02% buffered 3-aminobenzoic acid ethyl ester (tricaine, Sigma).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
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 dye (GE Healthcare) and column purified.
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| |
|
| Channel 2 |
| Source name |
D.rerio_embryo_PBS 24hpi B
|
| Organism |
Danio rerio |
| Characteristics |
PBS-injected zebrafish embryos at 24 hours post injection
|
| Biomaterial provider |
Oliver Stockhammer, IBL, Leiden University
|
| Treatment protocol |
All injection experiments were performed using mixed egg clutches from three tanks of AB strain zebrafish. Embryos were staged at 27 hours post fertilization (hpf) by morphological criteria (Kimmel et al., 1995) and a volume of PBS was injected into the caudal vein close to the urogenital opening.
|
| 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 -30 °C in egg water (60µg/ml Instant Ocean sea salts) For the duration of PBS injections embryos were kept under anaesthesia in egg water containing 0.02% buffered 3-aminobenzoic acid ethyl ester (tricaine, Sigma). Embryos were harvested 24 hours post injection.
|
| 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 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_embryo_CR vs PBS 24hpi_replica B
|
| 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. Processed data were subsequently imported into Rosetta Resolver 7.0 (Rosetta Biosoftware, Seattle, Washington) and subjected to default ratio error modelling. The fold change was calculated by dividing the test intensity value by the reference value. If this number was less than one the (negative) reciprocal is listed.
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| |
|
| Submission date |
Dec 16, 2008 |
| Last update date |
Dec 17, 2008 |
| 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) |
| GSE13994 |
Salmonella typhimurium infection of zebrafish embryo |
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