High-throughput sequencing and analysis of microbial community cDNA (metatranscriptomics) are providing valuable insight into in situ microbial activity and metabolism in the oceans. A critical first step in metatranscriptomic studies is the preparation of high-quality cDNA. At the minimum, preparing cDNA for sequencing involves steps of biomass collection, RNA preservation, total RNA extraction, and cDNA synthesis. Each of these steps may present unique challenges for marine microbial samples, particularly for deep-sea samples whose transcriptional profiles may change between water collection and RNA preservation. Because bacterioplankton community RNA yields may be relatively low (<500ng), it is often necessary to amplify total RNA to obtain sufficient cDNA for downstream sequencing. Additionally, depending on the nature of the samples, budgetary considerations, and the choice of sequencing technology, steps may be required to deplete the amount of ribosomal RNA (rRNA) transcripts in a sample in order to maximize mRNA recovery. cDNA preparation may also involve the addition of internal RNA standards to biomass samples, thereby allowing for absolute quantification of transcript abundance following sequencing.
This chapter describes a general protocol for cDNA preparation from planktonic microbial communities, from RNA preservation to final cDNA synthesis, with specific emphasis placed on topics of sampling bias and rRNA depletion. Consideration of these topics is critical for helping standardize metatranscriptomics methods as they become widespread in marine microbiology research.
- Stewart FJ. (2013) Preparation of microbial community cDNA for metatranscriptomic analysis in marine plankton. Methods Enzymol 531,187-218. [abstract]