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Technical considerations for RNA‐based stable isotope probing: an approach to associating microbial diversity with microbial community function
Author(s) -
Manefield Mike,
Whiteley Andrew S.,
Ostle Nick,
Ineson Philip,
Bailey Mark J.
Publication year - 2002
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.782
Subject(s) - chemistry , stable isotope probing , rna , agarose gel electrophoresis , microbial population biology , temperature gradient gel electrophoresis , stable isotope ratio , isotope , isotope ratio mass spectrometry , agarose , environmental chemistry , chromatography , mass spectrometry , computational biology , biochemistry , gene , bacteria , genetics , biology , microorganism , physics , 16s ribosomal rna , quantum mechanics
An ongoing challenge within microbial ecology is the development of methodologies that attribute microbial community functions to microbial diversity. One approach, involving the incorporation of stable isotopes from labelled tracer compounds into biological signature molecules (biomarkers), may overcome this current limitation. To examine the potential of RNA as the biomarker in stable isotope probing we have generated a series of atom % 13 C‐enriched RNA samples through exploitation of the anabolic abilities of a phenol‐degrading environmental isolate. Isotope ratio mass spectrometry was used to determine the atom % 13 C of each RNA sample (ca. 1–100%). The corresponding buoyant density (1.755–1.795 g mL −1 ) was determined by equilibrium density gradient centrifugation and agarose gel electrophoresis. This empirically defined relationship between the atom % 13 C of RNA and its buoyant density suggests ribonucleic acids with atom % 13 C enrichments greater than 10% can be isolated by equilibrium density centrifugation. The processing and analysis of isolated RNA by reverse transcription polymerase chain reaction, denaturing gradient gel electrophoresis, cloning and sequencing are discussed. The RNA‐based stable isotope probing protocol presented here will find particular utility in assessing the roles of microbial community members in the biodegradation of natural and anthropogenic xenobiotic compounds. Copyright © 2002 John Wiley & Sons, Ltd.