Method paperHigh-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study
Introduction
Copepods are the most abundant multicellular organisms on the planet, with extraordinary diversity in their morphologies, physiologies and life-strategies, forming an important link between phytoplankton and fish in the ‘classic’ pelagic food web (Humes, 1994, Runge, 1988). Through their life processes and vertical migrations, they play an important role in carbon transfer to the deep ocean and thus contribute to biogeochemical cycling (Frangoulis et al., 2005). Ultimately, they are also an emerging model system for ecotoxicological and environmental genomics studies (Raisuddin et al., 2007). Despite their global abundance and ecological importance, however, very few large-scale genomic resources exist for copepods.
With the recent development of Next Generation Sequencing (NGS) platforms (e.g. Illumina, 454, and SOLiD), it is now possible to address specific ecological and evolutionary questions in non-model organisms using transcriptome sequencing (RNA-Seq) (Ekblom and Galindo, 2011, Hudson, 2008). Although, several RNA-Seq studies have been performed on calanoids (Lenz et al., 2014, Ning et al., 2013), no information on the quantity or quality of the extracted RNA was given, making it difficult to ascertain the true efficiency of the method for isolating RNA from copepods.
To date, no systematic comparison of preservation and RNA isolation protocols for copepod RNA-Seq has been reported. Since copepod transcriptomics is a growing field and more studies are expected in the near future, it is critical to define a standard and reliable total RNA extraction protocol. The aim of the present study was to perform a comparative analysis of two preservation reagents, TRIzol® and RNAlater®, and three RNA extraction methods: a GTPC separation method using TRIzol® reagent, and two widely-used Silica Membrane (SM) based commercial kits, Qiagen RNeasy Micro Kit (Qiagen) and Aurum Total RNA Mini Kit (BioRad), in the ubiquitous large-sized calanoid copepod Calanus helgolandicus to obtain high-quality total RNA for NGS. All methods were evaluated with regard to the quantity and quality of the isolated RNA, in terms of purity (measured as A260/280 and A260/230 ratios), and integrity (measured as RNA Integrity Number, RIN). After optimization on C. helgolandicus, the optimal protocol was also tested on the small-sized calanoid copepods Centropages typicus and Temora stylifera. Our results provide a standard protocol for isolating high quality copepod RNA for high-throughput NGS studies and will help increase the number of copepod genomic resources in the near future.
Section snippets
Sample collection
Zooplankton samples were collected weekly from April to May 2012 at a fixed coastal station in the Gulf of Naples (40.80°N, 14.25°E), with vertical hauls using a 200-μm mesh size plankton net, and samples were brought to the laboratory within 4 h. Live C. helgolandicus males and females (n = 100–200) were sorted from the sample under a stereomicroscope (Leica), kept for several days on a diet of the dinoflagellate Prorocentrum minimum and finally preserved for RNA extraction. Males and females of
Results and discussion
Overall, we performed 55 extractions of total RNA from C. helgolandicus. Results are summarized in Table 1. Total RNA quantity has been indicated separately for males and females, since significantly higher amounts of RNA were obtained from females than males (540.2 ± 403.6 ng/female vs. 224.9 ± 203.9 ng/male, respectively) (unpaired t-test, t = 3.831 df = 51, p < 0.001). This difference is probably related to the females' large body size and active metabolism during reproduction, as suggested by Zhang and
Conclusion
To our knowledge, this is the first systematic assessment of different protocols for extraction of total RNA from copepods which is suitable for Next Generation Sequencing technologies. We were able to optimize extraction of high quality and quantity RNA from the copepods C. helgolandicus, C. typicus and T. stylifera by a combination of whole-body preservation with RNAlater®, followed by homogenization using a micropestle, and extraction of total RNA using the Qiagen RNeasy Micro Kit.
Author contributions
S. A. and Y. C. designed and performed the experiments and drafted the manuscript. C. L. helped in RNA extraction and copepod sorting. Y. C. and A. I. conceived the study and revised the manuscript along with P.K.L. All authors have read and approved the final manuscript.
Additional information
The authors declare that they have no competing financial interests.
Acknowledgments
We thank the Servizio Pesca of the Stazione Zoologica Anton Dohrn for the zooplankton sampling and Francesco Esposito for algal stock maintenance and cultivation. We also thank Rita Marino for running RNA samples on Agilent Bioanalyzer. This research was funded by Stazione Zoologica Anton Dohrn S. A. has been supported by a Stazione Zoologica Anton Dohrn PhD fellowship.
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