title: Technical considerations for RNA-based stable isotope probing: an approach to associating microbial diversity with microbial community function.
abstract: 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.
authors.affiliation: CEH Oxford, Mansfield Road, Oxford OX1 3SR, UK.
authors.forename: Mike
authors.initials: M
authors.lastname: Manefield
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Whiteley
authors.forename: Nick
authors.initials: N
authors.lastname: Ostle
authors.forename: Philip
authors.initials: P
authors.lastname: Ineson
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Bailey
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002247:Carbon Isotopes / Q000378:metabolism; D013058:Mass Spectrometry; D019800:Phenol / Q000378:metabolism; D016958:Pseudomonas putida / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D015347:RNA Probes; D012329:RNA, Bacterial / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012988:Soil Microbiology
chemical_list: D015415:Biomarkers; D002247:Carbon Isotopes; D015347:RNA Probes; D012329:RNA, Bacterial; D019800:Phenol