Faculty of Medical and Life Sciences, Institute of Precision Medicine, Microbiology and Hygiene Group, Furtwangen University, Villingen-Schwenningen, Germany.
Institute of Applied Microbiology, Research Center for BioSystems, Land Use, and Nutrition (IFZ), Justus-Liebig-University Giessen, Germany.
Can J Microbiol. 2020 Aug;66(8):491-494. doi: 10.1139/cjm-2019-0612. Epub 2020 Mar 5.
RNA-based stable isotope probing (RNA-SIP) is used in molecular microbial ecology to link the identity of microorganisms in a complex community with the assimilation of a distinct substrate. The technique is highly dependent on a reliable separation of isotopic-labeled RNA from unlabeled RNA by isopycnic density gradient ultracentrifugation. Here we show that C-labeled and unlabeled RNA can be sufficiently separated by isopycnic ultracentrifugation even in the absence of formamide. However, a slightly lower starting density is needed to obtain a distribution pattern similar to that obtained when formamide was used. Hence, the commonly used addition of formamide to the centrifugation solution might not be needed to separate C-labeled RNA from unlabeled RNA, but this must be verified for more complex environmental mixtures of RNA. Clearly, an omission of formamide would increase the safety of RNA-SIP analyses.
基于 RNA 的稳定同位素探测（RNA-SIP）被用于分子微生物生态学，以将复杂群落中微生物的身份与特定底物的同化联系起来。该技术高度依赖于等密度梯度超速离心，通过等密度密度梯度超速离心将同位素标记的 RNA 与未标记的 RNA 可靠分离。在这里，我们表明，即使没有甲酰胺，C 标记的和未标记的 RNA 也可以通过等密度超速离心充分分离。然而，需要稍微降低起始密度才能获得与使用甲酰胺时相似的分布模式。因此，可能不需要将甲酰胺添加到离心溶液中以将 C 标记的 RNA 与未标记的 RNA 分离，但这必须针对更复杂的 RNA 环境混合物进行验证。显然，省略甲酰胺会增加 RNA-SIP 分析的安全性。
