Format

Send to

Choose Destination
Methods. 2019 Oct 3. pii: S1046-2023(18)30486-9. doi: 10.1016/j.ymeth.2019.09.013. [Epub ahead of print]

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

Author information

1
IRI Life Sciences, Humboldt University, Philippstrasse 13, 10115 Berlin, Germany.
2
IRI Life Sciences, Humboldt University, Philippstrasse 13, 10115 Berlin, Germany. Electronic address: benedikt.beckmann@iri-lifesciences.de.

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.

KEYWORDS:

Organic extraction; RBP; RNP; UV cross-linking

PMID:
31586594
DOI:
10.1016/j.ymeth.2019.09.013
Free full text

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center