|
| Status |
Public on May 29, 2009 |
| Title |
embryo treated with 0.33uM RA, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Danio embryos at 11hpf treated with 0.33uM RA from 6.5 hpf
|
| Organism |
Danio rerio |
| Characteristics |
tissue: dechorionated embryo
genotype: wt incross from AB/Wik hybrid parents
age: embryos at 11hpf
|
| Treatment protocol |
When embryos reached the appropriate stage (5.25 hours post fertilization (hpf) for AGN193109 (Allergan, Irvine, CA), DMSO (Sigma), and 6.5 hpf for atRA [all-trans Retinoic Acid] (Sigma), they were transferred to agarose-coated dishes containing the appropriate amount of drug or comparable solvent. Embryos used for the microarray analysis were treated with 0.33 mM atRA, 10 mM AGN 193109, 1 mM AGN 193109 or 2% DMSO, all diluted 1:50 from stock solutions in DMSO. At the end of treatment, embryos were rinsed once in embryo media prior to RNA isolation.
|
| Growth protocol |
Zebrafish embryos of the *ABxWIK hybrid background were maintained at 28.5˚C under standard conditions prior to treatment. Embryos were dechorionated enzymatically before treatment and were kept in agarose-coated dishes during treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAwiz extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
RNA samples were labeled using the Affymetrix recommended protocol which includes a first and second strand cDNA synthesis from total RNA, followed by in vitro transcription (IVT) using the ENZO IVT kit to incorporate biotin into the synthesized cRNA. The biotin-labeled cRNA was purified over a column and quality was checked using the bioanalyzer and spectrophotometer. 20 µg of biotin-labeled cRNA was then fragmented, 15 µg was reserved for the hybridization and the remainder was used in quality control analysis on the bioanalyzer.
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| |
|
| Hybridization protocol |
Hybridization was performed utilizing the Affymetrix recommended protocol. 15 µg of fragmented biotin-labeled cRNA was combined with hybridization buffer. 200 l (10 µg) of this mix was then placed on the Zebrafish array and hybridized 16 hours at 45ºC at 60 rpm in a GeneChip Hybridization Oven 640. The arrays were washed and stained with Streptavidin Phycoerythrin (SAPE) using Affymetrix recommended protocols and a fluidics station 450. The wash protocol used was EukGE_WS2v4_450.
|
| Scan protocol |
The arrays were scanned using Affymetrix recommended protocols and a GeneChip Scanner 3000 with GCOS 1.2 software.
|
| Description |
Gene expression data from embryos in slow phase of cellularisation.
|
| Data processing |
All sample labeling and hybridizations, as well as chip processing and imaging were performed with strict adherence to Affymetrix’s standardized protocols in the FHCRC microarray core facility. A single cycle of target synthesis and labeling was used for all samples. Array normalization was performed using GCRMA (Wu et. al. 2004). Significant changes in expression level were identified using CyberT (Baldi and Long, 2001), a Bayesian t-statistic derived for microarray analysis. Significance was determined by ranking the Bayesian p-values and using a false discovery rate methodology (FDR) to account for multiple testing (Benjamini, 1995). A FDR of 5% was used for the analysis.
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| |
|
| Submission date |
May 28, 2009 |
| Last update date |
May 28, 2009 |
| Contact name |
Lei Feng |
| E-mail(s) |
fengl@u.washington.edu
|
| Phone |
206-667-5697
|
| Fax |
206-667-5432
|
| Organization name |
FHCRC
|
| Department |
Basic Science
|
| Lab |
Moens
|
| Street address |
1100 Fairview Ave. North, B2-152
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL1319 |
| Series (1) |
| GSE16264 |
Zebrafish embryos treated with Retinoic Acid or Retinoic acid antagonist during gastrulation and early somitogenesis. |
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