Ghori Noor-Ul-Huda, Moreira-Grez Benjamin, Vuong Paton, Waite Ian, Morald Tim, Wise Michael, Whiteley Andrew S
Molecular Microbial Ecology Group, The UWA School of Agriculture and Enviornment (SAgE), The University of Western Australia, Crawley, WA, Australia.
Department of Computer Science and Engineering, The University of Western Australia, Perth, WA, Australia.
Methods Mol Biol. 2019;2046:31-44. doi: 10.1007/978-1-4939-9721-3_3.
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.
稳定同位素探测是一种结合分子和同位素的技术，用于探测环境样品中未培养微生物的身份和功能。利用碳和氮等常见元素的稳定同位素，RNA-SIP利用了在向群落中脉冲注入标记底物后，细胞利用这些底物时，由于活跃代谢以及较重质量同位素掺入RNA而导致RNA浮力密度增加的原理。随后通过密度梯度离心将标记的RNA与未标记的RNA分离，接着通过测序鉴定RNA。因此，RNA稳定同位素探测是一种不依赖培养的技术，可同时提供有关微生物群落、组成和功能的信息。本章介绍了进行RNA-SIP实验的详细方案，包括从环境样品中提取的稳定同位素标记RNA的形成、超速离心和分级分析。
