title: High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study.
abstract: Despite the ecological importance of copepods, few Next Generation Sequencing studies (NGS) have been performed on small crustaceans, and a standard method for RNA extraction is lacking. In this study, we compared three commonly-used methods: TRIzol®, Aurum Total RNA Mini Kit and Qiagen RNeasy Micro Kit, in combination with preservation reagents TRIzol® or RNAlater®, to obtain high-quality and quantity of RNA from copepods for NGS. Total RNA was extracted from the copepods Calanus helgolandicus, Centropages typicus and Temora stylifera and its quantity and quality were evaluated using NanoDrop, agarose gel electrophoresis and Agilent Bioanalyzer. Our results demonstrate that preservation of copepods in RNAlater® and extraction with Qiagen RNeasy Micro Kit were the optimal isolation method for high-quality and quantity of RNA for NGS studies of C. helgolandicus. Intriguingly, C. helgolandicus 28S rRNA is formed by two subunits that separate after heat-denaturation and migrate along with 18S rRNA. This unique property of protostome RNA has never been reported in copepods. Overall, our comparative study on RNA extraction protocols will help increase gene expression studies on copepods using high-throughput applications, such as RNA-Seq and microarrays.
authors.affiliation: Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy. Electronic address: sneha.asai@gmail.com.
authors.forename: Sneha
authors.initials: S
authors.lastname: Asai
authors.affiliation: Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy.
authors.forename: Adrianna
authors.initials: A
authors.lastname: Ianora
authors.affiliation: Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Lauritano
authors.affiliation: Plymouth Marine Laboratory, Prospect Place, Plymouth PL1 3DH, UK.
authors.forename: Penelope K
authors.initials: PK
authors.lastname: Lindeque
authors.affiliation: Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy. Electronic address: ylenia@szn.it.
authors.forename: Ylenia
authors.initials: Y
authors.lastname: Carotenuto
mesh_terms: D000818:Animals; D033342:Copepoda / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D013045:Species Specificity; D013048:Specimen Handling / Q000379:methods*
chemical_list: D012337:RNA, Ribosomal, 18S
keywords: Copepods; NGS; RIN; Total RNA extraction; Transcriptome