Technische Universität München, Chair of Soil Ecology, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
J Microbiol Methods. 2011 Mar;84(3):406-12. doi: 10.1016/j.mimet.2010.12.028. Epub 2011 Jan 21.
We developed an improved protocol, allowing the simultaneous extraction of DNA and RNA from soil using phenol-chloroform with subsequent column-based separation of DNA and RNA (PCS). We compared this new approach with the well established protocol published by Griffiths et al. (2000), where DNA and RNA are separated by selective enzymatic digestions and two commercial kits used for DNA or RNA extraction, respectively, using four different agricultural soils. We compared yield and purity of the nucleic acids as well as abundance and diversity profiles of the soil bacterial communities targeting the nosZ gene via quantitative real-time PCR and terminal restriction fragment length polymorphism on DNA and RNA level. The newly developed protocol provided purer nucleic acid extracts compared to the used kit-based protocols. All protocols were suitable for DNA- and RNA-based gene quantification, however high variations between replicates were obtained for RNA samples using the original Griffiths protocol. Diversity patterns of nosZ were highly influenced by the extraction protocol used both on the DNA and RNA level. Finally, our data showed that the new protocol allows a simultaneous and reproducible extraction and separation of DNA and RNA, which were suitable for reliable analyses of gene and transcript copy numbers and diversity pattern.
我们开发了一种改良的方案，使用酚-氯仿同时从土壤中提取 DNA 和 RNA，随后通过基于柱的方法分离 DNA 和 RNA（PCS）。我们将这种新方法与 Griffiths 等人（2000 年）发表的成熟方案进行了比较，后者通过选择性酶消化和两种分别用于 DNA 或 RNA 提取的商业试剂盒在四种不同的农业土壤中分离 DNA 和 RNA。我们比较了核酸的产量和纯度，以及通过定量实时 PCR 和 DNA 和 RNA 水平上的末端限制性片段长度多态性靶向 nosZ 基因的土壤细菌群落的丰度和多样性图谱。与使用的基于试剂盒的方案相比，新开发的方案提供了更纯净的核酸提取物。所有方案都适用于 DNA 和 RNA 基基因定量，但使用原始 Griffiths 方案时，RNA 样品的重复间差异很大。nosZ 的多样性模式受到 DNA 和 RNA 水平上使用的提取方案的强烈影响。最后，我们的数据表明，新方案允许 DNA 和 RNA 的同时和可重复提取和分离，适用于基因和转录本拷贝数和多样性模式的可靠分析。
