Project description:Gene expression analyses have been performed on brain tissue of sexually mature and immature males using microarrays. 60 eels were transferred to two independent temperature controlled recirculation water units connected to two 500 L cylindro-conical tanks (30 fish per tank) where the fish were acclimated to seawater (35 PSU salinity) over a 2 week period.Eel males in one of the seawater units were injected intramuscularly every week over a 140 day period with 2000 IU hCG/kg (human chorionic gonadotropin, Sigma–Aldrich Chemical) dissolved in 0.9% saline to induce sexual maturation. Eel males in the other recirculation unit were injected weekly over the same period with 0.9% NaCl (vehicle).At the end of the experiment, eels were anesthetized in a solution of 0.1mg/L tricaine methanesulfonate (MS-222, Sigma Aldrich) and blood samples collected into heparinized syringes. Tissues (brain, including the olfactory bulbs, telencephalon, diencephalon and mesencephalon, and gonads) were collected from sexually immature (n=12) and sexually mature males (n=12).RNA was extracted from brain tissue of a total of 24 (12 mature and 12 immature) males. For each individual, RNA was extracted separately for olfactory bulb, telencephalon and diencephalon. Because RNA concentrations of olfactory bulb, telencephalon and diencephalon were below the amount needed for microarray analysis, a separate experiment for each part of the brain was not possible. Therefore, we combined equal concentrations of olfactory bulb, telencephalon and diencephalon as a single brain sample for each individual.Microarray analysis was conducted using an European eel-specific array consisting of a total of 14,913 probes based on a large collection of high-throughput transcriptomic sequences (Pujolar et al. 2012). Probe sequences and further details on the microarray platform can be found on the GEO database under accession number GPL15124. Data was normalized using a quantile normalization procedure using R (http://www.r-project.org)

Project description:Gene expression analyses have been performed on brain tissue of sexually mature and immature males using microarrays. 60 eels were transferred to two independent temperature controlled recirculation water units connected to two 500 L cylindro-conical tanks (30 fish per tank) where the fish were acclimated to seawater (35 PSU salinity) over a 2 week period.Eel males in one of the seawater units were injected intramuscularly every week over a 140 day period with 2000 IU hCG/kg (human chorionic gonadotropin, Sigma–Aldrich Chemical) dissolved in 0.9% saline to induce sexual maturation. Eel males in the other recirculation unit were injected weekly over the same period with 0.9% NaCl (vehicle).At the end of the experiment, eels were anesthetized in a solution of 0.1mg/L tricaine methanesulfonate (MS-222, Sigma Aldrich) and blood samples collected into heparinized syringes. Tissues (brain, including the olfactory bulbs, telencephalon, diencephalon and mesencephalon, and gonads) were collected from sexually immature (n=12) and sexually mature males (n=12).RNA was extracted from brain tissue of a total of 24 (12 mature and 12 immature) males. For each individual, RNA was extracted separately for olfactory bulb, telencephalon and diencephalon. Because RNA concentrations of olfactory bulb, telencephalon and diencephalon were below the amount needed for microarray analysis, a separate experiment for each part of the brain was not possible. Therefore, we combined equal concentrations of olfactory bulb, telencephalon and diencephalon as a single brain sample for each individual.Microarray analysis was conducted using an European eel-specific array consisting of a total of 14,913 probes based on a large collection of high-throughput transcriptomic sequences (Pujolar et al. 2012). Probe sequences and further details on the microarray platform can be found on the GEO database under accession number GPL15124. Data was normalized using a quantile normalization procedure using R (http://www.r-project.org) A comparative analysis of gene expression was conducted between sexually mature and immature males. Sample labelling and hybridization were conducted following the details in Pujolar et al. (2012). Hybridized slides were scanned at 5 µm resolution using an Agilent DNA microarray scanner. Slides were scanned at two different sensitivity levels (XDR Hi 100% and XDR Lo 10%) and the two linked images generated were analysed together. Data were extracted and background subtracted using the standard procedure in Agilent Feature Extraction (FE) software v. 9.5.1. Hybridization success was evaluated using flag values, excluding those intensities not equal to 1.

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The acute transcriptome response of the midbrain/diencephalon to injury in the adult mummichog (Fundulus heteroclitus)

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