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Sample GSM686155 Query DataSets for GSM686155
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.
 
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

Data table header descriptions
ID_REF
VALUE log2 normalized ratio Cy3/Cy5

Data table
ID_REF VALUE
5285955 -0.457383203
5285943 -1.685724358
5285859 0.366044059
5285847 0.19482259
5285763 0.129888276
5285751 0.952973954
5285667 -0.376807212
5285655 -0.091010752
5285571 0.183013647
5285559 0.082471571
5285475 -0.159639583
5285463 0.432174508
5285379 0.047677745
5285367 0.034061894
5285283 0.075188851
5285271 -0.124961937
5285187 0.047010037
5285175 -0.220249438
5285091 -0.162401504
5285079 -2.619001065

Total number of rows: 16416

Table truncated, full table size 319 Kbytes.




Supplementary file Size Download File type/resource
GSM686155.gpr.gz 1.4 Mb (ftp)(http) GPR
Processed data included within Sample table

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