Wild G. holbrooki were captured from one site downstream of the Buckeye Pulp and Paper Mill (Taylor County, Perry, FL, USA) on the Fenholloway River (GPS coordinates: N 30 058.341’, W 83 588.569’) and from one site in the Econfina conservation area (GPS coordinates: N 30 08.549', W 83 51.962'). One sampling event at the paper mill site was conducted on July 12th, 2012 and two sampling events at Econfina on August 7th and October 4th, 2012. Sexually mature female G. holbrooki were transferred to 5 gallon aerated buckets filled with site water and were processed at the site immediately after collection. Fish were anesthetized using Tricaine-S (Western Chemical, Ferndale, USA) and sacrificed via spinal transection. Livers were removed and stored in RNALater (Qiagen, Hilden, Germany) overnight at 4C before transferred for long-term storage at -80C.
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 were 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) and a specific activity > 8 was the cut-off value for further processing.
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 Scanner using one color scan setting for 8x15k array slides.
Description
Econ_L3 Gene expression of fish residing at clean reference site.
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) to obtain background subtracted Processed Signal intensities. Normalization was conducted using the LOESS method, and values are reported as Log2 transformed data.
