Soil metatranscriptomics

We explored the global transcriptome, or metatranscriptome, of soil microbial communities using the oxic surface layer and the anoxic bulk soil of flooded paddy soil as the model system. Using conventional Sanger sequencing, our first attempt to examine global gene expression allowed us to detect the active components of the microbial communities but provided limited insight into their ecosystem functions [Shrestha et al. (2009) Environ. Microbiol. 11: 960-970].

More recently, we developed an efficient method for extracting high quality mRNA from soil. Key steps in the isolation of total RNA are low-pH extraction (pH 5.0) and Q-Sepharose chromatography. The removal efficiency of humic acids was 94 to 98% for all soils tested. To enrich mRNA, subtractive hybridization of rRNA is most efficient. The rRNA-depleted RNA is of sufficient purity and integrity (size range of 0.3 to 4 kb) for further applications [Mettel et al. (2010) Appl. Environ. Microbiol. 76: 5995-6000].

The procedural steps of our current metatranscriptomic approaches involve (i) extraction of total RNA, (ii) enrichment of mRNA, (iii) cDNA synthesis and next-generation sequencing (454 pyrosequencing, Illumina HiSeq/MiSeq), (iv) preprocessing of raw reads, and (v) data analysis using either online tools (rRNA: QIIME, Mothur, Silva; mRNA: Camera, MG-Rast) or a local bioinformatic pipeline [5].

Reference

[5] Kim, Y., Wegner, C.E., Liesack, W. (2014) Soil Metatranscriptomics, p. 63-93. In P. Nannipieri, G. Pietramellara, and G. Renell (ed.), Omics in Soil Science, Caister Academic Press.

Go to Editor View