|
| Status |
Public on Oct 01, 2011 |
| Title |
Embryo PBS injected rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
D.rerio_embryo_CR
|
| Organism |
Danio rerio |
| Characteristics |
comment: 24 hpf embryos were manually dechorionated and directly after onset of bloodflow injected in the caudal vein with 200 CFUs of E. tarda or with PBS. At 8 hpi the embryos were quenched in liquid nitrogen and stored at -80˚C. This sample is a mixture of embryos from the same experiment but not used as a sample on the microarray.
sample type: pooled common reference
|
| Treatment protocol |
Zebrafish were dechorionated manually at 24 hpf. After onset of bloodflow embryos were injected with 200 CFUs of E. tarda or with sterile PBS (phosphate buffered saline). At 8 hpi the embryos were quenched in liquid nitrogen and stored at -80°C.
|
| 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 were individually snap-frozen in liquid nitrogen and subsequently stored at -80°C. A frozen embryo was crushed with a chilled pestle and homogenized in 300 µl of TRI reagent (Ambion). 60 µl chloroform was added and the mixture was transferred to a 1.5 ml reaction tube containing 50 mg phase lock gel (Eppendorf) and incubated at room temperature for 5 minutes. The mixture was centrifuged at 12000 g at 4°C for 15 minutes, after which the aqueous phase was transferred to a fresh tube. 1 volume of 70 % ethanol was added and the mixture transferred to a RNeasy MinElute Cleanup kit (Qiagen) column which was centrifuged 15 seconds at 8000 g. 500 µl RPE buffer from the kit was applied to the column and centrifuged 15 seconds at 8000 g. 500 µl 80 % ethanol was applied to the column and centrifuged 2 minutes at 8000 g. The collection tube was replaced and the column centrifuged 5 minutes at 14000 g. 14 µl H2O was applied to the column and centrifuged 1 minute at 14000 g. The average amount of RNA isolated from a single embryo was 500 ng.
|
| Label |
Cy3
|
| Label protocol |
Amino Allyl modified aRNA was synthesized in one amplification round from 500 ng 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_non-infected_8hpi_1
|
| Organism |
Danio rerio |
| Characteristics |
tissue: embryo
time point: 36 hpi
infection: none
tissue: embryo
|
| Treatment protocol |
Zebrafish were dechorionated manually at 24 hpf. After onset of bloodflow embryos were injected with 200 CFUs of E. tarda or with sterile PBS (phosphate buffered saline). At 8 hpi the embryos were quenched in liquid nitrogen and stored at -80°C.
|
| 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 were individually snap-frozen in liquid nitrogen and subsequently stored at -80°C. A frozen embryo was crushed with a chilled pestle and homogenized in 300 µl of TRI reagent (Ambion). 60 µl chloroform was added and the mixture was transferred to a 1.5 ml reaction tube containing 50 mg phase lock gel (Eppendorf) and incubated at room temperature for 5 minutes. The mixture was centrifuged at 12000 g at 4°C for 15 minutes, after which the aqueous phase was transferred to a fresh tube. 1 volume of 70 % ethanol was added and the mixture transferred to a RNeasy MinElute Cleanup kit (Qiagen) column which was centrifuged 15 seconds at 8000 g. 500 µl RPE buffer from the kit was applied to the column and centrifuged 15 seconds at 8000 g. 500 µl 80 % ethanol was applied to the column and centrifuged 2 minutes at 8000 g. The collection tube was replaced and the column centrifuged 5 minutes at 14000 g. 14 µl H2O was applied to the column and centrifuged 1 minute at 14000 g. The average amount of RNA isolated from a single embryo was 500 ng.
|
| Label |
Cy5
|
| Label protocol |
Amino Allyl modified aRNA was synthesized in one amplification round from 500 ng 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 |
825 ng for each channel were combined and made to 55 μl with 10x Blocking Agent and 25x Fragmentation Buffer. 55 μl of 2x Hi-RPM Hybridization Buffer was added (Gene Expression Hybridization Kit, Agilent Technologies). 100 μl of hybridization mixture was loaded onto each array and allowed to hybridize at 65°C (10 RPM) for 17h. The slides were washed with Gene Expression Wash Buffers I and II (Gene Expression Wash Buffer Kit, Agilent Technologies).
|
| Scan protocol |
Slides were scanned on an Agilent G2505B scanner at 5 μm resolution and data was extracted with Feature Extraction version 9.3.1.1. (Protocol GE2-v5_95).
|
| Description |
The CR (common reference) was composed by combining RNA of excess embryos from the caudal vein injection experiment
Biological replicate 1 of 3: Zebrafish embryos injected in the caudal vein with phosphate buffered saline (PBS)
|
| 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.
|
| |
|
| 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 |
GPL13390 |
| Series (2) |
| GSE28485 |
Transcriptome analysis of the zebrafish embryonic host response to Edwardsiella tarda infection [experiment B] |
| GSE28486 |
Transcriptome analysis of the zebrafish embryonic host response to Edwardsiella tarda infection |
|
