Rna later solution

Pituitary, ovary and testes tissues were harvested and placed immediately into RNAlater solution (Life Technologies). Pancreatic islets were picked from pancreatic tissue digested following direct intra pancreatic injection of 2 mL of 0.2 mg/mL Liberase (Roche) and placed immediately into RNAlater solution (25 (link)). Total RNA was extracted from the tissues using the RNeasy kit (Qiagen), and up to 1µg of total RNA was used to generate cDNA using the Quantitect RT kit (Qiagen), as described (26 (link)). Quantitect primers (Qiagen) were used for qRT-PCR reactions, which utilised the Quantitect SYBR green kit (Qiagen), on a RotorGene 5 (Qiagen), as described (26 (link)). Each test sample was normalized to the geometric mean of reference genes GAPDH, calnexin and α-tubulin. The relative expression of target cDNA in all qRT-PCR studies was determined using the Pfaffl method (27 (link)).

Adult E. nipponicum were collected from C. carpio caught in the littoral zone of the Mušov lowland reservoir (48°53′12″N, 16°34′37″E; Czech Republic). Soluble worm extract and excretory-secretory products were prepared as described previously^{5 (link)}, whereby the protein concentration was determined using the Quaint-iT^TM Protein Assay Kit (Life Technologies) and a SpectraMax i3 fluorometer (Molecular Devices). Samples of the E. nipponicum tissue for isolating the RNA were immersed in RNAlater^® Solution (Roche). All samples were stored in −80 °C.
Ethics statement: All procedures performed in studies involving animals were carried out in accordance with European Directive 2010/63/EU and Czech laws 246/1992 and 359/2012 which regulate research involving animals. All experiments were performed with the legal consent of the Animal Care and Use Committee of Masaryk University and of the Research and Development Section of the Ministry of Education, Youth, and Sports of the Czech Republic.