Gene Expression Analysis in Fish Gonads

Gene expression analysis was performed on fish at five stages: recrudescence (n = 5), developing (n = 6), pre-spawning (n = 7), spawning (n = 7), and post-spawning (n = 6) (n = 31 microarrays). Microarray hybridizations were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol using Cyanine 3 (Cy3) and 100 ng total RNA per sample was used for the production of cDNA and labeled/amplified cRNA as per the Agilent Low RNA Input Fluorescent Amplification Kit. Labeling methods followed those previously described [18 (link)]. Each RBD testis sample showed a specific activity >6.0 pmol Cy3/mL and amounts were adjusted to a final mass of 0.825 μg for 8x15K microarray hybridizations. Fragmentation of the cRNA, hybridization, and slide washes after the 17 h hybridization followed instructions outlined by Agilent (Gene Expression Hybridization Kit). An ozone barrier slide was used to cover microarrays before scanning. Microarrays were scanned at 5 μm with the high density Agilent DNA Microarray Scanner (Agilent Technologies). Agilent Feature Extraction Software (v9.5) scanned arrays with a full dynamic range. The quality of microarray data was evaluated by manual inspection and the quality control report generated by Feature Extraction Software (Agilent Technologies). Each microarray was deemed to be of high quality based on visual inspection.