|
| Status |
Public on Mar 08, 2011 |
| Title |
Zebrafish exposed to Estrogen 3 (17b-estradiol; 10mM final concentration) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Zebrafish exposed to Estrogen (17b-estradiol; 10mM final concentration)
|
| Organism |
Danio rerio |
| Characteristics |
treatment group: E2 treatment
|
| Treatment protocol |
A batch of healthy adult males were exposed to the medium containing 17β-estradiol (E2; Sigma-Aldrich; 10 nM final concentration) and an equal number of males were exposed to the medium containing a combination of E2 (10 nM) and ICI 182,780 (Tocris Cookson) (1 µM final concentration). Control fishes (males) were maintained in water containing 0.01% (v/v) ethanol (ethanol control; ethanol was used to dissolve E2 and ICI). The final ethanol concentration in the control was similar to those in medium with E2 or E2 and ICI.
Minimum 3 replicates were maintained for each treatment. Control and experimental animals were maintained in their respective medium for 96 hours and the medium was changed every day during the course of the experiment. Upon completion of the experiment at 96 hours, the fish were snapped frozen in liquid nitrogen and stored at -80°C for subsequent analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from individual frozen fish belonging to control and experimental samples (ethanol control, E2 and E2+ICI treated). Frozen samples were homogenized to a crude powder form in a pre-cooled mortar and pestle. Partially homogenized sample was transferred to a pre-cooled sterile graduated falcon tube containing appropriate amount of Trizol reagent (Invitrogen, USA) and homogenized completely using a motorized homogenizer. Total RNA was extracted from the samples using Trizol reagent according to manufacturer’s instructions. Subsequently RNA was purified using Qiagen column and the quality was evaluated using gel electrophoresis
|
| Label |
Cy3
|
| Label protocol |
Sample and reference RNA were reverse transcribed in the presence of Cy3-dUTP and Cy5-dUTP (Amersham Inc.), respectively, to fluorescently label the target cDNAs.
|
| |
|
| Channel 2 |
| Source name |
Reference (control)
|
| Organism |
Danio rerio |
| Characteristics |
treatment group: reference
|
| Treatment protocol |
A batch of healthy adult males were exposed to the medium containing 17β-estradiol (E2; Sigma-Aldrich; 10 nM final concentration) and an equal number of males were exposed to the medium containing a combination of E2 (10 nM) and ICI 182,780 (Tocris Cookson) (1 µM final concentration). Control fishes (males) were maintained in water containing 0.01% (v/v) ethanol (ethanol control; ethanol was used to dissolve E2 and ICI). The final ethanol concentration in the control was similar to those in medium with E2 or E2 and ICI.
Minimum 3 replicates were maintained for each treatment. Control and experimental animals were maintained in their respective medium for 96 hours and the medium was changed every day during the course of the experiment. Upon completion of the experiment at 96 hours, the fish were snapped frozen in liquid nitrogen and stored at -80°C for subsequent analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from individual frozen fish belonging to control and experimental samples (ethanol control, E2 and E2+ICI treated). Frozen samples were homogenized to a crude powder form in a pre-cooled mortar and pestle. Partially homogenized sample was transferred to a pre-cooled sterile graduated falcon tube containing appropriate amount of Trizol reagent (Invitrogen, USA) and homogenized completely using a motorized homogenizer. Total RNA was extracted from the samples using Trizol reagent according to manufacturer’s instructions. Subsequently RNA was purified using Qiagen column and the quality was evaluated using gel electrophoresis
|
| Label |
Cy5
|
| Label protocol |
Sample and reference RNA were reverse transcribed in the presence of Cy3-dUTP and Cy5-dUTP (Amersham Inc.), respectively, to fluorescently label the target cDNAs.
|
| |
|
| |
| Hybridization protocol |
The arrays were hybridized following the strategies described in. A minimum of three good hybridizations were selected for the analysis.
|
| Scan protocol |
The signal intensities of Cy5 and Cy3 dyes in each spot and the local background were measured using the GenePix 4000B microarray scanner (Axon Instruments, USA) to calculate the net intensity of each spot for analysis.
|
| Description |
replicate 3
|
| Data processing |
Compugen microarray set (Compugen, USA) containing 16,416 oligonucleotide probes representing zebrafish genes was used in this study. Briefly, the oligonucleotide probes were spotted onto poly-L-lysine coated microscope slides using a custom-built DNA microarrayer and post-processed following the standard procedures previously described.
Microarray data from GenePix image analysis software (i.e gpr files) were subjected to Lowess normalization. There were 4 control (ethanol controls) samples, 3 samples treated with E2 and 3 samples treated with E2 and ICI in the normalized data, respectively. The good arrays were selected based on scatter plot analysis.
Significance Analysis of Microarrays (SAM) was used to identify the prominent signals for the two kinds of treatments as previously described. To boost the power of the test, we applied 3-class SAM on the whole array by excluding spots with more than 6 missing values. As a result, 1610 out of 16416 genes were selected at q-value <8%. Subsequently, estrogen-responsive genes were identified by analyzing the expression values for the samples treated with E2 and E2+ICI with respect to control samples using 2-class SAM. The genes selected following this analysis displayed between 2- to 130-fold differential expressions. A cluster of 715 up-regulated genes were identified which showed significant up regulation in E2. These genes displayed decreased expression both in the Control and E2+ICI compared to their level of expression in E2. Similarly, another cluster of 376 genes (down-regulated group) were identified which displayed significantly reduced expression in E2 compared to control and E2+ICI. Hence, the genes selected by the above analysis are estrogen-responsive and are sensitive to E2 and ICI. Datasets extracted using the above statistical analysis were clustered and visualized as previously described.
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| |
|
| Submission date |
Mar 07, 2011 |
| Last update date |
Mar 08, 2011 |
| Contact name |
Justin Jeyakani Joseph Gnanakkan |
| E-mail(s) |
gnanakkan@gis.a-star.edu.sg
|
| Phone |
68088173
|
| Organization name |
Genome Institute of Singapore
|
| Department |
Computational and Systems Biology
|
| Street address |
60, Biopolis street
|
| City |
Singapore |
| State/province |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL2715 |
| Series (1) |
| GSE27707 |
Zebrafish: Estrogen induced gene expression |
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