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.
Growth protocol
G. holbrooki were captured using pole nets from a pristine river in a New South Wales (NSW) national park
Extracted molecule
total RNA
Extraction protocol
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).
Label
Cy3
Label protocol
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 protocol
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).
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scannerusing one color scan setting for 8x15k array slides.
Description
Gene expression after 48 hour exposure to levonorgestrel (100 ng/L)
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) to obtain background subtracted Processed Signal intensities.
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
