High-quality RNA extraction from the sea urchin <i>Paracentrotus lividus</i> embryos

<div><p>The sea urchin <i>Paracentrotus lividus</i> (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. <i>P</i>. <i>lividus</i> is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from <i>P</i>. <i>lividus</i> embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this established model marine organism.</p></div

Dose-dependent variation of gene expression levels induced by decadienal for the genes analyzed.

<p>Histograms show dose-dependent variations in expression levels of decadienal modulated genes. Samples incubated with increasing decadienal concentrations (1.0, 1.3, 1.6, 2.0, 2.3 μM) were collected at different stages of development: early blastula (5hp), swimming blastula (9hpf), prism (24hpf) and pluteus (48 hpf) Data are reported as a fold difference (mean ± SD), compared to the control embryos in sea water without decadienal. Fold differences greater than ±2 (see the dotted horizontal guide lines at the values of +2 and −2) were considered significant. A colour code has been used in the histograms to distinguish the four functional classes of genes: green for stress genes, grey for genes involved in detoxification processes, blue for genes involved in developmental and differentiation processes, and red for skeletogenic genes.</p

Gene expression level of HUB genes.

<p>The histograms show the differences in the expression levels of the four HUB genes followed by real-time qPCR, identified in <i>P</i>. <i>lividus</i>: <i>NF-κB</i>, <i>δ-2-catenin</i>, <i>HIF1A</i> and <i>p53</i>. Embryos were grown in the presence of decadienal, heptadienal and octadienal at teratogenic concentrations (1.6, 3.0 and 4.5 μM, respectively) and collected at different times of development (5, 9, 24 and 48hpf). Data are reported as a fold difference (mean ± SD), compared to the control, embryos in sea water without aldehydes. Fold differences greater than ±2 (see the dotted horizontal guide lines at the values of +2 and −2) were considered significant.</p

Interactomic analysis by Ingenuity Pathway Analysis (IPA) software.

<p>The network is displayed graphically as nodes (genes) and edges (the biological relationships between nodes). HUB nodes, genes that share the largest numbers of connections with other genes, are indicated by symbols of different colors: <i>RELA</i> in green; <i>CTNNB1</i> in red, <i>HIF1A</i> in light blue and <i>TP53</i> in yellow. The biological relationships between HUB nodes and the other significant genes are indicated by coloured arrows (indicating that a molecule modulates the expression of another), according to the colours of the HUB to which they are connected. The connections between CTNNB1-HIF1A, CTNNB1-RELA are indicated by edges and not by arrowheads because the solid edges indicate direct relationships between molecules due to real chemical modifications and, hence, to formation of direct physical contacts. Interaction between HUB nodes are indicated with grey arrows. Genes associated with HUB genes are reported with grey symbols. For further details on IPA analysis see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149734#sec002" target="_blank">Materials and Methods</a> section.</p

Agilent Bioanlyzer electropherograms.

<p>Examples of representative Agilent Bioanlyzer electropherograms of <i>P</i>. <i>lividus</i> RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172171#pone.0172171.t001" target="_blank">Table 1</a>). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.</p

Working model of hypothetical pathways potentially involved in the toxic PUAs stress response.

<p>PUAs may be able to activate four HUB genes (δ-2-catenin regulates <i>NF-κB</i>, which regulates <i>HIF1A</i>, which in its turn is regulated by <i>p53</i>; a crosstalk between <i>p53</i> and <i>NF-κB</i> has been reported in ref. 41) or several genes (belonging to different functional responses). All these genes may induce teratogenesis and/or to apoptosis in the sea urchin embryo.</p

Synopsis of dose-dependent gene expression of genes analyzed.

<p>Patterns of dose-dependent up- (red arrows) and down-regulation (blue arrows) of the four classes of genes in the sea urchin, <i>P. lividus</i>, in the presence of the PUAs decadienal, heptadienal and octadienal. Genes with two arrows are up- and down-regulated at different concentrations. The arrows correspond to fold differences greater than ±2, considered significant levels of down- or up-regulation.</p

Gene functional classes.

<p>The scheme indicates the four functional classes of genes used to which the thirty one genes analyzed in the present study belong: canonical stress genes, genes involved in detoxification processes, genes involved in developmental and differentiation processes and skeletogenic genes.</p

The corresponding names of <i>P</i>. <i>lividus</i> and human genes are reported.

<p>The corresponding names of <i>P</i>. <i>lividus</i> and human genes are reported.</p

Accession numbers, primer sequences and lengths of PCR amplified fragments are reported for the four new genes analyzed.

<p>Accession numbers, primer sequences and lengths of PCR amplified fragments are reported for the four new genes analyzed.</p