Comment[ArrayExpressAccession]	E-GEOD-55518
MAGE-TAB Version	1.1						
Public Release Date	2014-09-01
Investigation Title	Hepatic transcriptome analysis of male and female Eastern Mosquitofish (Gambusia holbrooki) exposed to a progestin and anti-progesterone reveals the mode of action of endocrine disruption of synthetic steroids in an aquatic organism						
Comment[Submitted Name]	Hepatic transcriptome analysis of male and female Eastern Mosquitofish (Gambusia holbrooki) exposed to a progestin and anti-progesterone reveals the mode of action of endocrine disruption of synthetic steroids in an aquatic organism
Experiment Description	Major classes of hormone mimics that have been studied include environmental estrogens and androgens, but recent studies have also demonstrated the significant impacts of natural and synthetic progesterones in the environment. The objective of this study was to evaluate the molecular and physiological impacts of progestin, anti-progestin, and mixture exposures in the Eastern Mosquitofish (G. holbrooki). By comparison of gene expression profiles and modulated biological processes in the three groups, it was determined that mifepristone acts more as a progestin than as an anti-progestin, as has also been demonstrated in other species of fish. This work contributes to the overall knowledge of the impacts of this class of chemical contaminats on aquatic organisms, which are a sentinel species for pollutants as aquatic ecosystems often become a reservoir for anthropogenic contaminants. G. holbrooki adult males and females were exposed to one of the following conditions: vehicle control (ethanol), 100 ng/L of levonorgestrel, 100 ng/L of mifepristone, or a mixture of both levonorgestrel and mifepristone. All exposures were conducted for 48 hours with water changed and chemicals renewed daily. Fish were anesthetized using 100 mg/L Benzocaine (Ethyl 4-aminobenzoate). Livers were removed and stored in RNAlater (Qiagen, Hilden, Germany) overnight at 4 C before storage at -80 C. RNA was isolated from the livers using TRIzol (Invitrogen, Grand Island, USA), hydrated using RNAsecure (Ambion, Grand Island, USA), and DNase treated using the Turbo DNA-free kit (Ambion, Grand Island, USA). Four oocyte-development stage-matched RNA samples per treatment were evaluated for RNA integrity using the 2100 BioAnalyzer (Agilent, Santa Clara, USA). The range of RIN values was 8.2-9.6						
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl					
Person Last Name	Brockmeier	Erica	Nancy				
Person First Name	Erica	Brockmeier	Denslow				
Person Mid Initials	Karin						
Person Email	ericakarin13@gmail.com						
Person Affiliation	University of Florida						
Person Address	University of Florida, 2187 Mowry Road, Gainesville, FL, USA						
Person Roles	submitter						
Protocol Name	P-GSE55518-1	P-GSE55518-5	P-GSE55518-6	P-GSE55518-2	P-GSE55518-3	P-GSE55518-4	P-GSE55518-7
Protocol Description	The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) to obtain background subtracted Processed Signal intensities. ID_REF =  VALUE = normalized signal intensity	The Low RNA Input Amplification Kit for one color (Cy3) (Agilent, Santa Clara, USA) was used to synthesize cRNA. 200 ng RNA and RNA spike-in controls were incubated with T7 primer mix and cDNA synthesis immediately proceeded primer annealing. In vitro transcription and Cy3 incorporation was then conducted and purification of Cy3-labeled cRNA was completed using the RNeasy kit (QIAGEN, Hilden, Germany). cRNA concentrations and specific activities were determined by the Nanodrop (ThermoScientific, Waltham, USA).	Hybridization of 600 ng purified cRNA was completed with the Gene Expression hybridization kit (Agilent, Santa Clara, USA). Cy3-labeled cRNA was incubated with blocking agent and fragmentation buffer at 60 C for 30 minutes. Hybridization buffer was then added to the sample and 40 μL of the sample was added to the gasket. The microarray slide was incubated with the gasket for 17 hours at 65 C while spinning at 10 rpm. The slide was then washed and dried, and scanning was conducted at the University of Florida gene expression core by the Microarray Scanner (Agilent, Santa Clara, USA).	G. holbrooki adult males and females were exposed to one of the following conditions: vehicle control (ethanol), 100 ng/L of levonorgestrel, 100 ng/L of mifepristone, or a mixture of both levonorgestrel and mifepristone. All exposures were conducted for 48 hours with water changed and chemicals renewed daily.	G. holbrooki were captured using pole nets from a pristine river in a New South Wales (NSW) national park	Liver samples in RNALater were thawed on ice, blotted dry of excess RNAlater, and homogenized in 1mL of TRIzol (Invitrogen, Grand Island, USA). After precipitation in isopropanol and washing with ethanol, RNA was rehydrated with RNAsecure Reagent (Ambion, Grand Island, USA). RNA was DNase treated using Turbo DNase (Ambion, Grand Island, USA). RNA quality and quantity was assessed using the Nanodrop (ThermoScientific, Waltham, USA) and the 2100 BioAnalyzer (Agilent, Santa Clara, USA).	Slides were scanned immediately after washing on the Agilent DNA Microarray Scannerusing one color scan setting for 8x15k array slides.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	TREATMENT	SEX					
Experimental Factor Type	treatment	sex					
Comment[SecondaryAccession]	GSE55518						
Comment[GEOReleaseDate]	2014-09-01						
Comment[ArrayExpressSubmissionDate]	2014-03-03						
Comment[GEOLastUpdateDate]	2014-09-02						
Comment[AEExperimentType]	transcription profiling by array						
SDRF File	E-GEOD-55518.sdrf.txt