Evaluating methods for the preservation and extraction of DNA and RNA for analysis of microbial communities in marine sponges

Abstract

Preservation and extraction of high quality DNA and RNA from biological samples are becoming increasingly important for genomics, transcriptomics and microbial community analyses. Of major recent interest, due to their ecological and biotechnological importance, are the communities of microorganisms associated with marine sponges. In this study, we systematically evaluated protocols for the preservation (RNA later, liquid nitrogen, lyophilized, frozen) and extraction (CTAB-based DNA extraction, RNA isolation via TRIzol and two co-extraction methods) of nucleic acids using samples of the New Zealand high-microbial-abundance sponge Ancorina alata. The quantity and quality of nucleic acids were assessed via spectrophotometry, agarose gel electrophoresis and microcapillary electrophoresis. Although all protocols resulted in sufficiently high DNA and/or RNA quantity and quality for downstream applications, there were significant differences in yield of nucleic acids extracted. All RNA extraction methods maintained mRNA of sufficient integrity to amplify the prokaryotic glutamine synthetase gene. Denaturing gradient gel electrophoresis (DGGE) analysis of community 16S rRNA gene- and 16S rRNA-derived fragments revealed several unique bands in the rRNA-derived profiles. However, there were no major changes attributable to either preservation or extraction method. Optimized methods were successfully tested on three other New Zealand sponges. Our results suggest that whilst choice of preservation and extraction method does influence the quantity of nucleic acids isolated from marine sponges, all protocols performed favorably, therefore the choice of protocol can be made based on practical considerations including ease of use, time availability and cost.