RNA stable-isotope probing

Abstract

At the heart of microbial ecology lies a true scientific dichotomy. On the one hand, we know microbes are responsible for processes on which all other life on Earth is dependent; their removal would mean the cessation of all known life. However, in opposition, the majority of extant microbial species in natural environments have never been cultured or studied in a laboratory as living organisms. Owing to these factors, the question of “who does what?” has been a major barrier to understanding how microbially mediated ecosystem level events occur. Recently, the use of stable isotopes (13C) to trace carbon from specific substrates into microbes that assimilate carbon from that substrate has significantly advanced our understanding of the relationship between environmental processes and microbial phylogeny.

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Figure 1: Typical total nucleic acid extract followed by Qiagen Allprep RNA/DNA column purification.
Figure 2: Typical gradient fraction analysis (100 μl fractions) by density of a 2.2 ml volume 1.8 g ml−1 starting density CsTFA gradient after 46 h of centrifugation.

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Acknowledgements

A.S.W. acknowledges Dr Ian Douglas of Beckman Coulter for his continuous expert input into the centrifugation equipment technologies required during RNA-SIP development. We acknowledge The UK's Natural Environment Research Council for financial support to develop RNA-SIP. T.L. acknowledges the Helmholtz Society for funding during the preparation of this manuscript. We finally thank two anonymous referees whose comments provided additional clarifications to the protocol.

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Correspondence to Andrew S Whiteley.

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Whiteley, A., Thomson, B., Lueders, T. et al. RNA stable-isotope probing. Nat Protoc 2, 838–844 (2007). https://doi.org/10.1038/nprot.2007.115

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