Rnaqueous midi kit purification system

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).