|
| Status |
Public on Aug 01, 2013 |
| Title |
zebrafish mycobacterium yolk injection embryos experiment 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
D.rerio_infected_experiment 2
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: 5 days post fertilisation embryos
agent: Mycobacterium marinum
|
| Treatment protocol |
Zebrafish embryos were either untreated or micro-injected into the yolk (2hpf) with 12 CFU (+/- 7) of Mycobacterium strain E11 mCherry bacteria suspended in PVP (Polyvinylpyrrolidone), At 5 days post fertilization embryos were snap frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent.
|
| 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_control
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: 5 days post fertilisation embryos
agent: none (control)
|
| Treatment protocol |
Zebrafish embryos were either untreated or micro-injected into the yolk (2hpf) with 12 CFU (+/- 7) of Mycobacterium strain E11 mCherry bacteria suspended in PVP (Polyvinylpyrrolidone), At 5 days post fertilization embryos were snap frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent.
|
| 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 the Microarray Department (MAD) of the University of Amsterdam (Amsterdam, 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 |
Single sample
|
| 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. The Agilent Feature Extraction files contain the final log ratio data. The algorithms that were used to normalize the feature signal and calculate the log ratios are described in the Reference Guide for Agilent Feature Extraction Software v9.5. The standard settings as described for the GE2-v5_95 protocol in the Reference Guide were used. In order to calculate the normalized signal, the Rank Consistency Probes method was used and the Linear&LOWESSDyeNormFactor (= DyeNormalSignal/(BGSubSignal x LinearDyeNormFactor) was calculated as described on page 231 of the Reference Guide. The dye normalized signal was calculated as follows (page 232): DyeNormSignal = BGSubSignal x DyeNormFactor. The log10 ratio was subsequently calculated as follows (page 232): LogRatio = Log(rProcessedSignal/gProcessedSignal), where rProcessedSignal and gProcessedSignal are signals post dye normalization and post surrogate processing in the red and green channels, respectively. The surrogate values are calculated and used as the lowest limit of detection to replace the dye normalized signal when either the mean feature signal is less than the background signal or not significant when compared to the background signal, or when the mean signal is less than its background standard deviation (page 226).
|
| |
|
| Submission date |
Feb 11, 2013 |
| Last update date |
Aug 01, 2013 |
| Contact name |
Herman Pieter Spaink |
| E-mail(s) |
h.p.spaink@biology.leidenuniv.nl
|
| Phone |
31715275055
|
| Organization name |
Leiden University
|
| Department |
Institute of Biology
|
| Lab |
Molecular Cell Biology
|
| Street address |
Einsteinweg 55
|
| City |
Leiden |
| ZIP/Postal code |
2333 CC |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL15180 |
| Series (2) |
| GSE44226 |
Gene expression profiling of zebrafish embryos at 5 days post fertilization [Agilent Arrays] |
| GSE44352 |
Gene expression profiling of zebrafish embryos at 5 days post fertilization |
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