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DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms

  • Eleanor Jameson
  • Martin Taubert
  • Sara Coyotzi
  • Yin Chen
  • Özge Eyice
  • Hendrik Schäfer
  • J. Colin Murrell
  • Josh D. Neufeld
  • Marc G. DumontEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1539)

Abstract

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter we provide protocols for obtaining labeled DNA, RNA, and proteins that can be used for downstream omics-based analyses.

Key words

Stable-isotope probing DNA RNA Protein Metagenomics Metatranscriptomics Proteomics 

Notes

Acknowledgments

Yin Chen acknowledges funding from NERC grant NE/I027061/1, and Yin Chen and J. Colin Murrell both acknowledge funding from the Gordon and Betty Moore Foundation Marine Microbiology Initiative Grant GBMF3303 and the Earth and Life Systems Alliance, Norwich Research Park, Norwich, UK. Josh D. Neufeld acknowledges a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Eleanor Jameson
    • 1
  • Martin Taubert
    • 2
  • Sara Coyotzi
    • 3
  • Yin Chen
    • 1
  • Özge Eyice
    • 4
  • Hendrik Schäfer
    • 1
  • J. Colin Murrell
    • 5
  • Josh D. Neufeld
    • 3
  • Marc G. Dumont
    • 6
    Email author
  1. 1.School of Life SciencesUniversity of WarwickCoventryUK
  2. 2.Institute of EcologyFriedrich Schiller University JenaJenaGermany
  3. 3.Department of BiologyUniversity of WaterlooWaterlooCanada
  4. 4.School of Biological and Chemical SciencesQueen Mary University of LondonLondonUK
  5. 5.School of Environmental SciencesUniversity of East AngliaNorwichUK
  6. 6.Centre for Biological SciencesUniversity of SouthamptonSouthamptonUK

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