Computational protocol: Genetic Inactivation of European Sea Bass (Dicentrarchus labrax L.) Eggs Using UV-Irradiation: Observations and Perspectives

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Protocol publication

[…] Extraction was performed on 250 mg freeze-dried sea bass eggs in a 15 ml cuvette with 5 ml of methanol/water (50/50, v/v) (analytical grade, Sigma-Aldrich, St-Quentin Fallavier, France), assisted by sonication during 10 min. The sample was then centrifuged 5 min at 10,000 rpm and the final extract diluted in Acetonitrile (ACN) (1/1, v/v) (analytical grade, Sigma-Aldrich, St-Quentin Fallavier, France).Ultra-high performance liquid chromatography (UHPLC) analyses were performed by a Dionex UltiMate 3000 RSLC system (Thermo Fisher Scientific, Waltham, USA) equipped for separation with a Kinetex HILIC (1.7 µm, 2.1×100 mm) (Phenomenex, Le Pecq, France) maintained at 40°C. The mobile phases consisted on (A) 10 mM Ammonium acetate (HPLC grade, Sigma-Aldrich, St-Quentin Fallavier, France) and (B) ACN (HPLC grade, Sigma-Aldrich, France) at constant flow-rate of 0.4 ml.min−1 (with gradient conditions described in ).Metabolic fingerprints were measured using a Dionex UltiMate 3000 RSLC system coupled to an AB SCIEX TripleTOF 560 quadrupole-time-of-flight mass spectrometer (AB SCIEX, Concord, ON, Canada). Mass-spectrometric analysis was performed using an electrospray ion source (ESI) in both positive and negative ion mode. In the positive ESI mode, parameters were: capillary voltage of 4500 V, nebulizing gas pressure of 60 psi, drying gas pressure of 60 psi, temperature of 550°C and declustering potential of 80 V. The capillary voltage in negative ESI was −4000 V and the other source settings were the same as for positive ESI. Information Dependent Acquisition (IDA) method was employed to collect MS and MS/MS accurate mass. TOF MS and TOF MS/MS were scanned with the mass range of m/z 80–1200.Instrument control and data acquisition were carried out with the Analyst 1.5.1 TF software (AB Sciex, Concord, ON, Canada) and the analysis was performed using Peak View 2.0 (AB Sciex, Concord, ON, Canada) also equipped with the MasterView Formula Finder and directly linked to ChemSpider database. [...] At all stages, survival was estimated as a percentage of developing eggs over the total number of eggs. Survival rates were calculated relative to controls after adjustment of the latter to 100%. Survival rates were arcsin square roots transformed for comparison between groups by two-way ANOVA using Statistica (Version 7.1). Data are presented as means ± standard deviations (STD). Presence of null alleles in the PCR products was analyzed using Microchecker software version 2.2.3 . […]

Pipeline specifications

Software tools MasterView, Statistica
Databases ChemSpider
Applications Miscellaneous, MS-based targeted metabolomics
Organisms Dipturus trachyderma, Dicentrarchus labrax