Identification of mirna-target rna interactions using clash

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

We present a detailed protocol for the experimental identifi cation of miRNA-target RNA interaction sites using c ross-linking, l igation, a nd s equencing of h ybrids (CLASH). The basis of the technique is the purifi cation of UV-stabilized Argonaute (AGO)–RNA complexes assembled in living cells, with subsequent ligation of AGO-associated RNA-RNA duplexes to form chimeric RNAs. Following cDNA synthesis, DNA library preparation and high-throughput sequencing, interacting RNA molecules are unambiguously identifi ed as chimeric reads in bioinformatic analysis of sequencing data. CLASH potentially recovers any RNA duplex that is bound by RNA-binding protein, so modifi ed approaches would be suitable for the identifi cation of many other inter- and intramolecular RNA-RNA interactions. Since CLASH analysis is independent of bioinformatic predictions it allows the identifi cation and analysis of RNA targeting rules in an unbiased way.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHUMANA PRESS INC
Pages229-251
Number of pages23
Volume1358
DOIs
Publication statusPublished - 1 Jan 2016

Publication series

NameMethods in Molecular Biology
Volume1358
ISSN (Print)1064-3745

Keywords

  • argonaute
  • CLASH
  • CLIP
  • MicroRNA
  • MiRNA target identifi cation
  • Protein-RNA interaction s
  • RNA cross-linking
  • RNA-RNA interactions
  • UV cross-linking

Fingerprint

Dive into the research topics of 'Identification of mirna-target rna interactions using clash'. Together they form a unique fingerprint.

Cite this