|
| Status |
Public on Jan 01, 2010 |
| Title |
TCDD exposed embryos, replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Universal RNA Reference Sample
|
| Organism |
Danio rerio |
| Characteristics |
Strain: TL
Age: 1-6 days post-fertilization
|
| Biomaterial provider |
M.E. Hahn (Woods Hole Oceanographic Institution)
|
| Treatment protocol |
Separate groups of 30 embryos generated from TL adults were placed in 15 ml egg water in a 10-cm glass petri and at 1, 2, 3, 4, 5, and 6-dpf were exposed for 6 hr to 0.1% dimethyl sulfoxide (DMSO), 2 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or 10 uM tert-butylhydroquinone (tBHQ) (4 groups per compound per time point). Embyros were frozen immediately after the 6 hr exposure and RNA isolated for microarray analysis. The Universal RNA Reference Sample was created by mixing equal amounts of total RNA from 2 replicates each from all toxicants (TCDD, tBHQ, DMSO) and timepoints (1, 2, 3, 4, 5, 6-dpf).
|
| Growth protocol |
Adult fish were maintained according to the guidelines set forth by the zebrafish handbook. Embryos were kept in egg water (60 μg/ml Instant Ocean Salts) in a 28°C incubator with no light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from pooled tissues using RNA STAT-60.
|
| Label |
Cy3
|
| Label protocol |
RNA samples were checked for quality using a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 BioAnalyzer. cDNA synthesis from 600 ng of total RNA was performed using the Agilent Low RNA Input Linear Fluorescent Amplification Plus kit, following the manufacturer’s instructions. cRNA was synthesized from the cDNA template, with incorporation of cyanine-3-CTP (Perkin Elmer). Labeled cRNA was purified using the Qiagen RNeasy Mini kit with quantity and quality was assessed by NanoDrop spectrophotometer and Agilent BioAnalyzer.
|
| |
|
| Channel 2 |
| Source name |
4 days post-fertilization TCDD exposed Danio rerio embryos, replicate 3
|
| Organism |
Danio rerio |
| Characteristics |
Strain: TL
Age: 4 days post-fertilization
|
| Biomaterial provider |
M.E. Hahn (Woods Hole Oceanographic Institution)
|
| Treatment protocol |
Separate groups of 30 embryos generated from TL adults were placed in 15 ml egg water in a 10-cm glass petri and at 4 dpf were exposed for 6 hr to 2 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Embyros were frozen immediately after the 6 hr exposure and RNA isolated for microarray analysis.
|
| Growth protocol |
Adult fish were maintained according to the guidelines set forth by the zebrafish handbook. Embryos were kept in egg water (60 μg/ml Instant Ocean Salts) in a 28°C incubator with no light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from pooled tissues using RNA STAT-60.
|
| Label |
Cy5
|
| Label protocol |
RNA samples were checked for quality using a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 BioAnalyzer. cDNA synthesis from 600 ng of total RNA was performed using the Agilent Low RNA Input Linear Fluorescent Amplification Plus kit, following the manufacturer’s instructions. cRNA was synthesized from the cDNA template, with incorporation of cyanine-5-CTP (Perkin Elmer). Labeled cRNA was purified using the Qiagen RNeasy Mini kit with quantity and quality was assessed by NanoDrop spectrophotometer and Agilent BioAnalyzer.
|
| |
|
| |
| Hybridization protocol |
cRNA samples were hybridized to Agilent 22k zebrafish microarrays using the Agilent Gene Expression Hybridization Kit. Labeled cRNAs were combined with the Agilent 25x fragmentation buffer and incubated at 60°C for 30 minutes. This was followed by mixing with 2x hybridization buffer, after which 100 ul of the product was spread evenly across the surface of an Agilent 22K zebrafish microarray. The loaded microarray was incubated at 60°C for 17 hours with rotation in an Agilent DNA Microarray Hybridization Oven. Post-hybridization, microarray slides were washed, air-dried, and stored in darkness with desiccation.
|
| Scan protocol |
Raw hybridization results were obtained by laser-excited fluorescence scanning in an Agilent DNA Micorarray Scanner.
|
| Description |
TCDD exposed embryos, replicate 3
|
| Data processing |
Analysis of raw microarray data was performed using Agilent’s feature extraction protocol, which includes spot finding, spot analysis, background subtraction (using local background plus global background based on spots along the central tendency line for red versus green intensity), dye normalization (linear and lowess algorithms, using spots along the central tendency line as for background subtraction), and final calculation of Cy5/Cy3 ratios and log2 transformed fold change for each spot. Data were additionally median centered among all microarray results prior to statistical analyses.
|
| |
|
| Submission date |
Jan 11, 2008 |
| Last update date |
Jun 24, 2009 |
| Contact name |
Andrew G. McArthur |
| E-mail(s) |
mcarthua@mcmaster.ca
|
| Organization name |
McMaster University
|
| Department |
Biochemistry & Biomedical Sciences
|
| Street address |
1280 Main Street West
|
| City |
Hamilton |
| State/province |
Ontario |
| ZIP/Postal code |
L8S 4K1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL2878 |
| Series (1) |
| GSE10157 |
Altered gene expression in zebrafish embryos exposed to tert-butylhydroquinone and 2,3,7,8-tetrachlorodibenzo-p-dioxin |
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