Isolation and analysis of mRNA from environmental microbial communities

Abstract

The advent of metagenomics has revealed that our planet harbors millions of previously undiscovered microbial species. However, functional insights into the activities of microbial communities cannot easily be obtained using metagenomics. Using transcriptional analyses to study microbial gene functions is currently problematic due to difficulties working with unstable microbial mRNA as a small fraction of total cellular RNA. Current techniques can be expensive and time consuming, and still result in significant levels of rRNA contamination. We have adapted techniques to rapidly isolate high high-quality RNA from environmental samples and developed a simple method for specific isolation of mRNA by size separation. This new technique was evaluated by constructing cDNA libraries directly from uncultured environmental microbial communities, including agricultural soil samples, aquatic flocculants, organic composts, mammalian oral and faecal samples, and wastewater sludge. The sequencing of a fraction of these cDNA clones revealed a high degree of novelty, demonstrating the potential of this approach to capture a large number of unique transcripts directly from the environment. To our knowledge, this is the first study that uses gel electrophoresis to isolate mRNA from microbial communities. We conclude that this method could be used to provide insights into the microbial ‘metatranscriptome’ of entire microbial communities. Coupled with high-throughput sequencing or the construction of cDNA microarrays, this approach will provide a useful tool to study the transcriptional activities of microorganisms, including those of entire microbial communities and of non-culturable microorganisms.