RNA Stable Isotope Probing (RNA-SIP)
Stable isotope probing is a combined molecular and isotopic technique used to probe the identity and function of uncultivated microorganisms within environmental samples. Employing stable isotopes of common elements such as carbon and nitrogen, RNA-SIP exploits an increase in the buoyant density of RNA caused by the active metabolism and incorporation of heavier mass isotopes into the RNA after cellular utilization of labeled substrates pulsed into the community. Labeled RNAs are subsequently separated from unlabeled RNAs by density gradient centrifugation followed by identification of the RNAs by sequencing. Therefore, RNA stable isotope probing is a culture-independent technique that provides simultaneous information about microbiome community, composition and function. This chapter presents the detailed protocol for performing an RNA-SIP experiment, including the formation, ultracentrifugation, and fractional analyses of stable isotope-labeled RNAs extracted from environmental samples.
Key wordsRNA-SIP 16S rRNA Gradient centrifugation Community diversity Community function Isotope-labeled substrate
The authors wish to thank all the investigators over the years who have played a role in developing RNA-SIP as a method, especially professors Mike Manefield, Tillmann Lüeders, and Dr. Ian Douglas of Beckman Coulter. Particular thanks go to Professor Colin Murrell for stimulating discussions over the years on stable isotope enhanced microbial ecology. The Molecular Microbial Ecology Laboratory at UWA is funded by a range of sources, including the Australian Research Council Linkage Program (LP150101111 to A.S.W.), The Australia China Joint Research Centre (A.S.W., I.W.), a Chilean BECAS scholarship (B.M.G.), and a UWA IPRS scholarship (N.H.G.).
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