RNA isolation from biological soil crusts: methodological aspects
RNA isolation from biological soil crusts: methodological aspects
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DOI：
10.1127/algol_stud/2016/0256
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发表时间：
2016-01-01
期刊：
影响因子：
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通讯作者：
Becker, Burkhard
中科院分区：
文献类型：
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作者：
Rippin, Martin;Komsic-Buchmann, Karin;Becker, Burkhard
生物土壤结皮（BSC）社区可以在几乎所有的环境中发现，除了热带地区。这些微生物垫在生态系统中尤其占主导地位，这些生态系统表现出恶劣的条件，它们作为初级生产者发挥着关键作用。研究它们的元转录组学数据使科学家能够阐明分类群的组成、这些生物之间的相互作用以及它们在极端环境（例如极地）中科普非生物压力的能力。成功的元转录组学分析的基础是高质量和完整性的纯RNA的分离。由于土壤的化学和物理性质不同，从土壤样品中提取核酸具有挑战性。腐殖物质通常被共同提取，随后污染样品。在这项研究中，测试和评价了不同的RNA提取技术，用于从四个BSC样品中分离高质量的RNA，三个在德国分离，一个极性BSC：基于CTAB的方案，Spectrum（TM）植物总RNA试剂盒和Precellys植物RNA试剂盒。基于CTAB的方法以低产量提供高质量的RNA，并且对于所有测试样品具有平均污染程度。使用Spectrum（TM）Plant Total RNA Kit获得的RNA具有高质量，只有很少的污染，但只有一半的样品可以成功处理。Precellys Plant RNA试剂盒是一种高通量方法，可获得良好的RNA质量，但污染水平最高。此外，四个样品中只有三个产生了任何结果。由于DNA和腐殖物质通常不容易去除，因此通常需要进一步纯化。使用酶消化，DNA可以被特异性地切割，而腐殖物质可以通过使用凝胶过滤或RNA亲和结合柱从RNA级分中分离。
Biological soil crust (BSC) communities can be found in almost all environments except for the tropics. These microbial mats are especially predominant in ecosystems, which exhibit harsh conditions, in which they play a key role as primary producers. Studying their metatranscriptomic data enables scientists to shed light on taxa composition, the interactions between those organisms and their ability to cope with abiotic stressors in the extreme environments, e.g. the polar regions. The basis of a successful metatranscriptomic analysis is the isolation of pure RNA of high quality and integrity. Nucleic acid extraction from soil samples is challenging due the diverse chemical and physical properties of soil. Humic substances are often co-extracted and, subsequently, contaminate the sample. In this study, different RNA extraction techniques were tested and evaluated for the isolation of high quality RNA from four BSC samples, three isolated in Germany and one polar BSC: a CTAB based protocol, the Spectrum (TM) Plant Total RNA Kit and the Precellys Plant RNA Kit. The CTAB based method provides high quality RNA at a low yield and with an average degree of contamination for all tested samples. RNA obtained by using the Spectrum (TM) Plant Total RNA Kit is of high quality with only little contamination but only half of the samples could be processed successfully. A high throughput approach is the Precellys Plant RNA Kit resulting in a fair RNA quality but with the highest level of contamination. Furthermore, only three out of four samples yielded any results. Further purification is often necessary as DNA and humic substances often cannot be easily removed. Using enzymatic digestion DNA can be specifically cleaved while humic substances can be separated from the RNA fraction by using either gel filtration or RNA affinity binding columns.