iCLIP

from Wikipedia, the free encyclopedia

iCLIP ( individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation , cross-linking and immunoprecipitation with single nucleotide resolution ') is a biochemical method for determining protein-RNA interactions . Such interactions take place e.g. B. with ribonucleoproteins and micro-RNA -containing protein complexes instead.

principle

The ICLIP uses a combination of different methods. After cross-linking of the RNA- protein complexes in cell cultures under UV light , the molecules bound to one another can be isolated together after a cell disruption due to the selective binding of the protein portion to existing antibodies in the course of an immunoprecipitation . The cross-linked RNA protein molecules are subjected to a marking of the RNA portion with radioactive phosphorus for later detection based on radioactivity . Following a done protein purification by SDS-PAGE with detection by Western blot . The complexes are extracted from the blot membrane in order to release the previously crosslinked RNA through a proteinase K digest of the protein components of the crosslinked complexes. The RNA is purified by phenol-chloroform extraction and used in an RT-PCR to generate cDNA with the same sequence of nucleobases . The cDNA is purified in a DNA polyacrylamide gel electrophoresis with subsequent gel extraction and ethanol precipitation . The purified DNA is by DNA sequencing in high-throughput sequencing. Since the determined sequences of the cDNA correspond to those of the RNA, the sequence of all RNA is determined that was previously also purified on the basis of the specific binding to a certain protein.

ICLIP has similarities with the CLIP-Seq , the PAR-CLIP , but with 64 reaction steps it is significantly more complex. In comparison, however, it can directly detect the cross-linked nucleotide. The SDS-PAGE provides additional protein purification , which reduces the amount of contaminant and undesired RNA in the RT-PCR that lead to the detection of false positive sequences.

Web links

  • starBase database : an online database for RNA-protein interactions. Retrieved June 20, 2013.
  • BIMSB doRiNA database : an online database for RNA-protein interactions. Retrieved June 20, 2013.

Individual evidence

  1. ↑ Julian König, Kathi Zarnack, Nicholas M. Luscombe, Jernej Ule: Protein – RNA interactions: new genomic technologies and perspectives . In: Nature Reviews Genetics . tape 13 , no. 2 , January 18, 2012, p. 77-83 , doi : 10.1038 / nrg3141 .
  2. ↑ JP Broughton, AE Pasquinelli: Identifying Argonaute binding sites in Caenorhabditis elegans using iCLIP. In: Methods. 63 (2), Sep 15, 2013, pp. 119-125. doi: 10.1016 / j.ymeth.2013.03.033 . PMID 23583680 .
  3. ^ J. Koenig, K. Zarnack, G. Rot, T. Curk, M. Kayikci, B. Zupan, DJ Turner, NM Luscombe, J. Ule: iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. In: Nature Structural Molecular Biology. Volume 17, Issue 7, 2010, pp. 909-915. doi: 10.1038 / nsmb.1838 . PMID 20601959 . PMC 3000544 (free full text).