Background: Microbes found on the skin (the skin microbiome) are important to skin function and health. Metatrancriptomics, the sequencing of RNA transcripts extracted directly from the environment, would provide insight into host-microbiome interactions. However the amounts of microbial RNA extracted from skin swabs are insufficient for the construction of RNA sequencing libraries. Amplification of this material is possible, but is often associated with bias.

Hypotheses: Verify the suitability of an amplification method previously used on a single prokaryotic cell for microarray analysis, for metatranscriptomics from limited quantities of a simulated skin microbial community.

Methods: Four skin isolates were mixed in equal ratios to simulate a skin microbiome community.  Optimization of cell lysis and RNA extraction were performed for high purity intact RNA. RNA was extracted from 10⁸, 10⁶ and 10⁴ cells of the simulated skin microbial community, and RT-PCR and amplification using Φ29 polymerase was performed.

Results: RNA quality was highly dependent in the cell disruption process.  High quality RNA was obtained from 10⁸ cells of the simulated skin microbial community, while poor quality RNA was obtained from lower numbers of cells. Amplification of cDNA from low cell numbers was yielded enough material for sequence library creation, but was extremely sensitive to DNA contamination.

Conclusion: RNA extraction from small numbers of cells requires further optimizes to yield high quality RNA. Amplification provides enough material for the generation of RNA sequencing libraries, but precautions must be taken to avoid all sources of contamination.  This method holds promise for the application of metatranscriptomics to the skin microbiome.