Rnalater tissue collection solution

In this study, the model organism Ciona robusta, was formerly classified as Ciona intestinalis. Molecular studies have confirmed that C. intestinalis constitutes a compilation of species rather than a single species [55 (link),56 (link),57 (link),58 (link)].
C. robusta specimens were collected from Sciacca harbor (Sicily, Italy) and were acclimatized and maintained as reported in Arizza et al. [33 (link)]
Fragments of pharynx tissue (200 mg) were immediately soaked in RNAlater tissue collection solution (AMBION, Austin, TX, USA) and Total RNA extraction was performed as reported in in Arizza et al. [33 (link)].

The animal model Ciona robusta was formerly classified as Ciona intestinalis. Molecular studies have confirmed that C. intestinalis constitutes a compilation of species rather than a single speciements [57 (link),58 (link),59 (link),60 (link),61 (link)]. C. robusta were collected from Sciacca harbour (Sicily, Italy) and were acclimatized and maintained as reported in Arizza et al. [34 (link)]. An LPS solution (Escherichia coli 055:B5, LPS, SIGMA-ALDRICH, Saint-Louis, MI, USA) was prepared in a sterile salt medium (12 mM CaCl2, 11 mM KCl, 26 mM MgCl2, 43 mM Tris HCl, 0.4 M NaCl, pH 8.0). One hundred microliters of the LPS-containing suspension was injected into the tunic matrix surrounding the pharynx wall (median body region) at a final LPS concentration of 100 μg. C. robusta not exposed to LPS (naïve) were used as controls. Fragments of pharynx tissue (200 mg) explanted at various times (from 1 to 48 h) and pharynx, ovary, intestine and stomach tissues of naïve were immediately soaked in RNAlater tissue collection solution (AMBION, Austin, TX, USA) and stored at −80 °C. Total RNA extraction was performed using an RNAqueous-Midi kit purification system (AMBION, Austin, TX, USA) as reported in Arizza et al. [34 (link)].

Molecular studies have led to the hypothesis that Ciona intestinalis constitutes a compilation of species rather than a single species^{10 (link)–13 (link)}. Accordingly, our model organism, originally from the Mediterranean Sea and formerly classified as C. intestinalis, corresponds to C. robusta^{10 (link)–13 (link)}.
C. robusta specimens were collected from Sciacca Harbour (Sicily, Italy) and were acclimatised and maintained under controlled temperature conditions (15 °C) in tanks supplied with flow-through oxygenated seawater. The animals were fed every 2 days with Coraliquid marine invertebrate food (Sera Heinsberg, Germany). An LPS solution (Escherichia coli 055:B5, LPS, SIGMA-ALDRICH, Germany) was prepared in sterile salt medium (12 mM CaCl₂, 11 mM KCl, 26 mM MgCl₂, 43 mM Tris HCl, 0.4 M NaCl, pH 8.0). One hundred microlitres of the LPS-containing suspension was injected into the tunic matrix surrounding the pharynx wall (median body region) at a final LPS concentration of 100 μg. C. robusta not exposed to LPS (naïve) were used as controls. Fragments of pharynx tissue (200 mg) explanted at various times (from 1 to 72 h) were immediately soaked in RNAlater tissue collection solution (AMBION, Austin, TX) and stored at − 80 °C. Total RNA extraction was performed using an RNAqueous-Midi kit purification system (AMBION, Austin, TX).