Sticky problems: extraction of nucleic acids from molluscs

Abstract

Traditional molecular methods and omics-techniques across molluscan taxonomy increasingly inform biology of Mollusca. Recovery of DNA and RNA for such studies is challenged by common biological properties of the highly diverse molluscs. Molluscan biomineralization, adhesive structures and mucus involve polyphenolic proteins and mucopolysaccharides that hinder DNA extraction or copurify to inhibit enzyme-catalysed molecular procedures. DNA extraction methods that employ the detergent hexadecyltrimethylammoniumbromide (CTAB) to remove these contaminants importantly facilitate molecular-level study of molluscs. Molluscan pigments may stain DNA samples and interfere with spectrophotometry, necessitating gel electrophoresis or fluorometry for accurate quantification. RNA can reliably be extracted but the 'hidden break' in 28S rRNA of molluscs (like most protostomes) causes 18S and 28S rRNA fragments to co-migrate electrophoretically. This challenges the standard quality control based on the ratio of 18S and 28S rRNA, developed for deuterostome animals. High-AT content in molluscan rRNA prevents the effective purification of polyadenylated mRNA. Awareness of these matters aids the continuous expansion of molecular malacology, enabling work also with museum specimens and next-generation sequencing, with the latter imposing unprecedented demands on DNA quality. Alternative methods to extract nucleic acids from molluscs are available from literature and, importantly, from communications with others who study the molecular biology of molluscs. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Keywords: AT rich; CTAB; hidden break; mollusca; mucopolysaccharides; pigment.

Figure 1.

DNA from molluscs is often intensely stained. Extraction of genomic DNA from the full body soft tissues of adult (Shell length about 8 mm) Physella acuta (Gastropoda, Heterobranchia, Hygrophila and Physidae), using a CTAB-based method according to Winnepenninckx et al. [37], routinely yields darkly stained DNA pellets (two replicate DNA samples precipitated in isopropanol and pelleted by centrifugation, shown on the right). The coloration is most likely owing to a pigment of unknown nature that co-purifies with the DNA. It is not a property of the DNA itself, or of a factor that routinely associates with the DNA: the staining is not present in DNA samples extracted from embryos (egg masses) of P. acuta using the same procedure (two replicate DNA pellet samples on the left). Both types of samples can be used for restriction digestion and PCR.

Figure 2.

Bioanalyzer profiles of two RNA samples of individual adult Physella acuta (Gastropoda, Heterobranchia, Hygrophila, Physidae). Following Trizol extraction from whole body soft tissues, 1 µl of Turbo DNA-free treated RNA analysed using the Agilent RNA 6000 Nano kit with Agilent Bioanalyzer software v. B.02.08.SI648(S2). Note the single rRNA peak (between 40 and 45 s), representing both 18S and similarly sized subunits 28S rRNA that dissociated during extraction due to the hidden break in 28S. The automatically calculated RIN (note low value) is ignored, rather the sample is interpreted to be of good quality based on showing well-defined, narrow peaks (20–45 s), especially the rRNA peak and low signal for larger sequences (greater than 50 s). (Online version in colour.)