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J Microbiol Methods. 2010 Jan;80(1):70-5. doi: 10.1016/j.mimet.2009.11.002. Epub 2009 Nov 10.
RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. (15)N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using (15)N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used (15)N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with (15)N-ammonium chloride or (15)N(2) we assessed the fractionation resolution of (15)N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between (15)N- and (14)N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments.
基于 RNA 的稳定同位素探测（SIP）有助于检测和鉴定参与同化同位素标记化合物的微生物种群中的活性成员。（15）N-RNA-SIP 是一种新方法，最近的文献中有讨论，但尚未经过测试。在此，我们定义了使用（15）N 标记底物进行 SIP 的限制，并提出了一些修改方法来弥补其中的一些缺点。我们已经使用（15）N-RNA-SIP 作为分析使用氮源底物的混合细菌种群的工具。在用（15）N-氯化铵或（15）N2 孵育混合微生物群落后，我们评估了等密度离心在三氟乙酸铯（CsTFA）梯度中的（15）N-RNA 分馏分辨率。我们发现，掺入的同位素标记越多，（15）N-RNA 和（14）N-RNA 之间的浮力密度（BD）分离就越远，但通过梯度分级分离不可能明确区分标记和未标记的 RNA。提取 RNA 的末端限制性片段长度多态性（T-RFLP）分析和富集培养物的荧光原位杂交（FISH）分析提供了一些关于固氮生物的信息。这种方法并非没有局限性，需要进一步发展以评估其在其他固氮环境中的适用性。
