Tissueruptor 2 homogenizer

Each fish lens was weighed prior to homogenization: for S. lucioperca, the typical lens weight was 100 mg, and for R. rutilus lacustris—40 mg. Only one lens from each fish was used for the analysis. The lens was placed in a glass vial and homogenized with a TissueRuptor II homogenizer (Qiagen, Netherlands) in 1600 µL of cold (−20 °C) MeOH, and then, 800 µL of water and 1600 µL of cold chloroform were added. The mixture was shaken well in a shaker for 20 min and left at −20 °C for 30 min. Then the mixture was centrifuged at 16,100× g, +4 °C for 30 min, yielding two immiscible liquid layers separated by a protein layer. The upper aqueous layer (MeOH-H₂O) was collected, divided into two parts for NMR (2/3) and LC-MS (1/3) analyses, and lyophilized.
Each fish gill was divided into arch and filaments. Only gill filaments were used for the analysis. Samples were weighed prior to homogenization: for S. lucioperca, the typical gill filament weight was 95 mg, and for R. rutilus lacustris—110 mg. The homogenization and extraction procedures for gill filaments were performed in the same way as for fish lenses.

The lens sample preparation was performed by the procedure that has been described in detail [36 (link),39 (link),40 (link)]. We analyzed either one lens per sample or pooled together two or three lenses from different individuals. Each sample was weighed prior to homogenization. The typical sample and lens weights are given in Table 1. Lenses were placed in glass vials and homogenized with a TissueRuptor II homogenizer (Qiagen, Netherlands) in 1600 µL of cold (−20 °C) MeOH, and then 800 µL of water and 1600 µL of cold chloroform were added. The mixture was shaken well in a shaker for 20 min and was left at −20 °C for 30 min. Then, the mixture was centrifuged at 16,100× g, +4 °C for 30 min, yielding two immiscible liquid layers separated by a protein layer. The upper aqueous layer (MeOH-H₂O) was collected, divided into two parts for NMR (2/3) and LC–MS (1/3) analyses and lyophilized.

At the experimental endpoint, mice were euthanized, and various tissues (spleen, liver, brain, placenta, and fetal head) were harvested and kept at −80°C until further use. Genomic DNA was extracted from 10 to 20 mg of tissue using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Indianapolis, IN, USA). Prior to extraction, tissues were disrupted in 400 μL of tissue lysis buffer using TissueRuptor II homogenizer (QIAGEN, Austin, TX, USA) at full speed for 30 sec. This homogenate was subjected to DNA extraction following the manufacturer’s recommendations. In the last step of the protocol, the DNA was eluted from the silica column in 100 μL of elution buffer, diluted 10 times before evaluation and stored at −20°C for further analysis.