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Fast and unbiased purification of RNA-protein complexes after UV cross-linking.

Authors
  • Urdaneta, Erika C1
  • Beckmann, Benedikt M2
  • 1 IRI Life Sciences, Humboldt University, Philippstrasse 13, 10115 Berlin, Germany. , (Germany)
  • 2 IRI Life Sciences, Humboldt University, Philippstrasse 13, 10115 Berlin, Germany. Electronic address: [email protected] , (Germany)
Type
Published Article
Journal
Methods
Publisher
Elsevier
Publication Date
Jun 01, 2020
Volume
178
Pages
72–82
Identifiers
DOI: 10.1016/j.ymeth.2019.09.013
PMID: 31586594
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

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. Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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