All fish sampled in the present study were adult female fathead minnows obtained from an on site culture facility at the US EPA Mid-Continent Ecology Division, Duluth, MN, USA.
Growth protocol
At the time of sample collection, fish were humanely killed in a buffered (200 mg Na2HCO3/L) solution tricaine methanesulfonate (MS-222; Finquel; Argent, Redmond WA, USA). Gentle pressure was applied to the abdomen of each fish. If eggs were released, the fish was noted to contain ovulated eggs, and the expressed oocytes were collected in a microcentrifuge tube and snap-frozen in liquid nitrogen. Blood was collected from the caudal peduncle using heparinized microcapillary tubes, and plasma was separated by centrifugation and subsequently stored at -80oC. Ovaries were removed and weighed. Tissue was transferred to a labeled microcentrifuge tube and snap-frozen in liquid nitrogen, then stored at -80o C until extracted.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the ovary or expelled oocyte (egg) samples using RNeasy kits (Qiagen, Valencia, CA, USA). RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent, Wilmington, DE, USA) and quantity was determined using a Nanodrop® ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Total RNA was stored at -80oC until analyzed.
Label
Cy3
Label protocol
cDNA synthesis, cRNA labelling, amplification and hybridization were performed following the manufacturer's kits and protocols (Quick Amp Labeling kit; Agilent, Palo Alto, CA).
Hybridization protocol
The Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies, Palo Alto, CA) was used for microarray hybridizations following the manufacturer’s protocol and recommendations.
Scan protocol
An Axon GenePix® 4000B Microarray Scanner (Molecular Devices Inc.) was used to scan microarray images at 5 μm resolution.
Description
Data were resolved from microarray images using Agilent Feature Extraction software (Agilent Technologies, Palo Alto, CA).
Data processing
Microarray image processing and data pre-processing were performed Agilent's Feature Extraction software v 9.5 following the manufacturer’s protocol and recommendations.
