DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.
DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.
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DOI：
10.1007/978-1-4939-6691-2_5
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发表时间：
2017
影响因子：
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通讯作者：
E. Jameson;M. Taubert;Sara Coyotzi;Yin Chen;Ö. Eyice;H. Schäfer;J. Murrell;J. Neufeld;M. Dumont
中科院分区：
文献类型：
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作者：
E. Jameson;M. Taubert;Sara Coyotzi;Yin Chen;Ö. Eyice;H. Schäfer;J. Murrell;J. Neufeld;M. Dumont
稳定同位素探测(SIP)使研究人员能够瞄准复杂微生物群落中的活跃种群，这是通过提供富含重同位素的生长底物实现的，重同位素通常以13C、18O或15N的形式存在。在底物上生长并随后提取微生物生物标记物(通常是核酸或蛋白质)后，使用SIP技术从活跃的微生物种群中回收和分析同位素标记的生物标记物。在基于DNA和RNA的SIP最初发展的几年里，通过靶向基因分析来表征标记的种群是一种常见的做法。这种方法通常包括基于指纹的分析或包含16S rRNA基因或功能标记基因扩增的克隆文库的测序。虽然分子指纹技术仍然是快速确认同位素标记的一种有价值的方法，但测序技术的最新进展意味着有可能从SIP实验中获得负担得起的和全面的扩增子谱、元基因组或元转录组。不仅可以从元基因组中推断微生物群体的丰富性，而且研究人员还可以将单个基因组进行绑定、组装和探索，以建立关于标记微生物代谢能力的假设。分析标记的mRNA是一项最新的进展，可以提供基于偏转录组的活性微生物的独立分析。后转录组学的力量在于，信使核糖核酸的丰度往往与相应的编码酶的活性密切相关，从而提供了对采样时微生物新陈代谢的洞察。总而言之，这些进展提高了SIP方法的灵敏度，并允许使用与生态相关的浓度的标记底物。特别是随着方法的改进和成本的不断下降，我们预计SIP与多种基于组学的方法的整合将成为微生物生态学研究的主流组成部分，从而在我们对新微生物种群的理解和阐明复杂微生物群落的代谢功能方面取得进一步突破。在本章中，我们提供了用于获得可用于下游基于组学的分析的标记DNA、RNA和蛋白质的协议。
Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of13C,18O, or15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter we provide protocols for obtaining labeled DNA, RNA, and proteins that can be used for downstream omics-based analyses.