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| Status |
Public on Aug 22, 2012 |
| Title |
MUS_96hr_maleBrain_4 |
| Sample type |
RNA |
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| Channel 1 |
| Source name |
brain_male_MUS_96 hr
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| Organism |
Danio rerio |
| Characteristics |
strain: AB
genotype/variation: wild-type
age: 5 months
gender: male
tissue: brain
treated with: MUS (muscimol) 500 ug/L for 96hr
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| Treatment protocol |
Reproductively mature male and female zebrafish were exposed to a continuous flow of test chemical (an analytically-confirmed concentration and a control), delivered in water (with no solvent), for 24, 48, or 96 hrs
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| Growth protocol |
Reproductively mature fish were obtained from an onsite culture unit at the US EPA National Health and Human Effects Research laboratory. Three days before the initiation of exposure, fish were randomly loaded into tanks receiving a continuous flow of Lake Superior water (USA) pumped from 200 m offshore at a depth of 20 m (mesh size 1 um, flow rate 45 ml/min). Each tank held 10 males and 10 females.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues were removed from RNAlater and placed on ice in 1.5-ml RNase-free microcentrifuge tubes (Ambion) containing 1 ml of TriReagent (Molecular Research Center,Cincinnati, OH)and then placed on ice for a short duration. They were homogenized by physical disruption at 30 Hz for 5 min in a Retsch MM300 mixer mill (Retsch Laboratory Equipment, Newtown, PA, USA). Individual homogenates were immediately processed or stored at 80C in TriReagent. Total RNA was extracted following the manufacturer's protocol. Briefly, 100 ul of 1-bromo-3-chloropropane (BCP, Molecular Research Center) were added to each homogenized tissue in 1 ml of TriReagent, vortexed, and incubated at room temperature for 10 min. The BCP/TriReagent mixture was then centrifuged at 12,000 g for 15 min at 4C. The aqueous phase was transferred to a new RNase-free microcentrifuge tube containing 500 ul of 100% isopropanol and then gently agitated. Precipitated samples were centrifuged at 12,000 g for 10 min at 4C, and supernatants were decanted. One milliliter of 75% ethanol was added to the precipitant, and samples were spot vortexed and centrifuged for 10 min at 12,000 g at 4C. Supernatant was decanted, and the remaining volume was aspirated. The RNA pellets were resuspended in nuclease-free water (Ambion) by incubation at 4C for several hours. To remove any trace organic contaminants, RNA samples were reprecipitated using a 0.1 volume of 3 M sodium acetate (Ambion) and two volumes of 100% ethanol and then incubating at 80C for at least 20 min. Precipitated RNA was centrifuged at 12,000 g at 4C for 20 min, and the supernatant was removed. The RNA was resuspended in RNase-free water at 4C for several hours. The RNA was then quantified spectrophotometrically, and light absorbance at 260 and 280 nm was used as a measure of relative purity and quantity. The RNA samples were analyzed for structural integrity using either agarose gel electrophoresis or the Bioanalyzer NanoRNA kit (Agilent Technologies, Palo Alto, CA, USA). All RNA displayed 260:280-nm ratios of 1.7 or greater and had 28S:18S ratios of approximately two. Extracted total RNA was stored at 80C until subsequent use.
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| Label |
Cy5
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| Label protocol |
Purified total RNA samples were shipped overnight on dryice to an Agilent-certified contract laboratory (Cogenics, Morrisville, NC 27560, USA), where all microarray gene expression profiling was performed. The quantity and quality of the RNA samples were assessed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies,Wilmington, DE, USA) and an Agilent 2100 bioanalyzer. One microgram of total RNA from each sample was amplified and labeled with a fluorescent dye (either Cy3 or Cy5) with the use of a low-RNA input linear amplification labeling kit following the manufacturer's protocol). The quantity and purity of the resulting fluorescently labeled cRNA was evaluated with the Nanodrop ND-1000 spectrophotometer, and the size distribution was determined with an Agilent bioanalyzer.
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| Channel 2 |
| Source name |
brain_male_water control_96 hr
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| Organism |
Danio rerio |
| Characteristics |
strain: AB
genotype/variation: wild-type
age: 5 months
gender: male
tissue: brain
treatment: in water control for 96 hr
|
| Treatment protocol |
Reproductively mature male and female zebrafish were exposed to a continuous flow of test chemical (an analytically-confirmed concentration and a control), delivered in water (with no solvent), for 24, 48, or 96 hrs
|
| Growth protocol |
Reproductively mature fish were obtained from an onsite culture unit at the US EPA National Health and Human Effects Research laboratory. Three days before the initiation of exposure, fish were randomly loaded into tanks receiving a continuous flow of Lake Superior water (USA) pumped from 200 m offshore at a depth of 20 m (mesh size 1 um, flow rate 45 ml/min). Each tank held 10 males and 10 females.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues were removed from RNAlater and placed on ice in 1.5-ml RNase-free microcentrifuge tubes (Ambion) containing 1 ml of TriReagent (Molecular Research Center,Cincinnati, OH)and then placed on ice for a short duration. They were homogenized by physical disruption at 30 Hz for 5 min in a Retsch MM300 mixer mill (Retsch Laboratory Equipment, Newtown, PA, USA). Individual homogenates were immediately processed or stored at 80C in TriReagent. Total RNA was extracted following the manufacturer's protocol. Briefly, 100 ul of 1-bromo-3-chloropropane (BCP, Molecular Research Center) were added to each homogenized tissue in 1 ml of TriReagent, vortexed, and incubated at room temperature for 10 min. The BCP/TriReagent mixture was then centrifuged at 12,000 g for 15 min at 4C. The aqueous phase was transferred to a new RNase-free microcentrifuge tube containing 500 ul of 100% isopropanol and then gently agitated. Precipitated samples were centrifuged at 12,000 g for 10 min at 4C, and supernatants were decanted. One milliliter of 75% ethanol was added to the precipitant, and samples were spot vortexed and centrifuged for 10 min at 12,000 g at 4C. Supernatant was decanted, and the remaining volume was aspirated. The RNA pellets were resuspended in nuclease-free water (Ambion) by incubation at 4C for several hours. To remove any trace organic contaminants, RNA samples were reprecipitated using a 0.1 volume of 3 M sodium acetate (Ambion) and two volumes of 100% ethanol and then incubating at 80C for at least 20 min. Precipitated RNA was centrifuged at 12,000 g at 4C for 20 min, and the supernatant was removed. The RNA was resuspended in RNase-free water at 4C for several hours. The RNA was then quantified spectrophotometrically, and light absorbance at 260 and 280 nm was used as a measure of relative purity and quantity. The RNA samples were analyzed for structural integrity using either agarose gel electrophoresis or the Bioanalyzer NanoRNA kit (Agilent Technologies, Palo Alto, CA, USA). All RNA displayed 260:280-nm ratios of 1.7 or greater and had 28S:18S ratios of approximately two. Extracted total RNA was stored at 80C until subsequent use.
|
| Label |
Cy3
|
| Label protocol |
Purified total RNA samples were shipped overnight on dryice to an Agilent-certified contract laboratory (Cogenics, Morrisville, NC 27560, USA), where all microarray gene expression profiling was performed. The quantity and quality of the RNA samples were assessed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies,Wilmington, DE, USA) and an Agilent 2100 bioanalyzer. One microgram of total RNA from each sample was amplified and labeled with a fluorescent dye (either Cy3 or Cy5) with the use of a low-RNA input linear amplification labeling kit following the manufacturer's protocol). The quantity and purity of the resulting fluorescently labeled cRNA was evaluated with the Nanodrop ND-1000 spectrophotometer, and the size distribution was determined with an Agilent bioanalyzer.
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| Hybridization protocol |
Equal amounts of Cy3- and Cy5-labeled cRNA (750 ng each) were hybridized to a zebrafish oligo microarray for 17 h at 65C. The hybridized microarrays were then washed according to the manufacturer's protocol.
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| Scan protocol |
Scanned by a 16-bit Agilent G2565BA scanner at 10-um resolution with the red and green photomultiplier tubes set to 100% and a scan area of 61 by 21.6 mm
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| Description |
Ave10294_1_4rProcessedSignal
Ave10294_1_4gProcessedSignal
for gene classifier discovery
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| Data processing |
Text output of individual samples (rProcessedSignal, gProcessedSignal) from Agilent Feature Extraction software were filtered according to Agilent recommendations (feature saturation, uniformity, pixel population consistency, signal strength relative to background; Agilent Technologies Training Manual, version 4.5). Samples were then organized either by tissue types or combined. Missing data were imputed by KNNimputer (Troyanskaya et al., 2001, Bioinformatics). This was followed by thresholding, log2 transformation, per chip normalization to 75th percentile, and per gene normation to the median of all samples [available as Series supplementary files].
The biological samples for controls were duplicated between TRI_96hr_femaleOvary and TRI_96hr_femaleOvaryLowDose as well as between TRI_96hr_maleTestis and TRI_96hr_maleTestisLowDose. These redundant controls were therefore included only once in their respective matrix files (classifier_discovery_ovary.txt, classifier_discovery_testis.txt), giving rise to their odd number of columns. In total, there are five processed/normalized matrix files either for classifier discovery or validation:
The numeric identifiers in the data matrix column headers (Sample Description field above) correspond to the unique microarray identifiers in the file names of raw text files. For example, rProcessedSignal'612' for US23502387_251322310'612'_S01_22k.txt, and Ave'10237_1_2'rProcessedSignal for US23502303_2515064'10237'_S01_GE2-v5_95_Feb07_'1_2'.txt
classifier_discovery_brain.txt: 80 treated + 80 control
classifier_discovery_ovary.txt: 105 treated + 100 control
classifier_discovery_testis.txt: 84 treated + 79 control
classifier_discovery_allTissue.txt: all three tissues combined plus 21 liver samples, 290 treated + 290 control
classifier_validation.txt: 24 treated + 28 control.
The processed/normalized data files contain rProcessedSignal and gProcessedSignal (log2 transformed, per chip and per gene normalized) from the indicated samples.
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| Submission date |
May 21, 2012 |
| Last update date |
Aug 22, 2012 |
| Contact name |
Rong-Lin Wang |
| E-mail(s) |
wang.rong-lin@epa.gov
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| Phone |
513-569-7862
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| Organization name |
US EPA
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| Street address |
26 W MLK Dr
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| City |
Cincinnati |
| ZIP/Postal code |
45268 |
| Country |
USA |
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| Platform ID |
GPL7302 |
| Series (1) |
| GSE38070 |
Discovery and validation of gene classifiers for endocrine disrupting chemicals in Zebrafish (Danio rerio) |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM933558_US23502303_251506410294_S01_GE2-v5_95_Feb07_1_4.txt.gz |
13.6 Mb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
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