IRI Life Sciences, Humboldt University, Philippstrasse 13, 10115 Berlin, Germany.
IRI Life Sciences, Humboldt University, Philippstrasse 13, 10115 Berlin, Germany.
Methods. 2020 Jun 1;178:72-82. doi: 10.1016/j.ymeth.2019.09.013. Epub 2019 Oct 3.
Post-transcriptional regulation of gene expression in cells is facilitated by formation of RNA-protein complexes (RNPs). While many methods to study eukaryotic (m)RNPs rely on purification of polyadenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we developed Phenol Toluol extraction (PTex), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 h) and simple protocol. The purification principle is solely based on physicochemical properties of cross-linked RNPs, enabling us to interrogate RNA-protein interactions system-wide and beyond poly(A) RNA from a variety of species and source material. Here, we are presenting an introduction of the underlying separation principles and give a detailed discussion of the individual steps as well as incorporation of PTex in high-throughput pipelines.
细胞中转录后基因表达的调控是通过 RNA-蛋白质复合物（RNP）的形成来实现的。虽然有许多研究真核（m）RNP 的方法依赖于多聚腺苷酸化 RNA 的纯化，但其他重要的调节 RNA 类或细菌 mRNA 则无法在同一深度进行研究。为了克服这一限制，我们开发了 Phenol Toluol extraction（PTex），这是一种新颖且无偏的在活细胞中纯化 UV 交联 RNP 的方法。PTex 是一种快速（2-3 小时）且简单的方案。纯化原理仅基于交联 RNP 的物理化学性质，使我们能够系统地研究 RNA-蛋白质相互作用，并且超越了来自多种物种和来源材料的 poly(A) RNA。在这里，我们将介绍基础分离原理，并详细讨论各个步骤以及将 PTex 纳入高通量管道。
