PAR CLIP

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PAR-CLIP ( Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation , 'cross-linking and immunoprecipitation with photoactivatable ribonucleotides') 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 PAR-CLIP is a combination of a UV - cross-linking , an immunoprecipitation , a partial nucleolysis , a RT-PCR and DNA sequencing in high throughput . The PAR-CLIP incorporates the UV-reactive nucleoside analogues 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) in vivo . Irradiation of the cells with a wavelength of 365 nm leads to cross-linking with the interacting proteins. This is followed by immunoprecipitation based on the known protein and RT-PCR of the isolated RNA. The DNA sequences of the generated cDNA is determined by DNA sequencing.

The PAR-CLIP has similarities to the CLIP-Seq and the iCLIP , which manage without nucleoside analogues. The RIP chip uses a microarray instead of RT-PCR and DNA sequencing. The ChIP-Seq is used to study protein-DNA interactions and the ChIP-on-Chip is used to study protein-DNA interactions with a microarray instead of DNA sequencing.

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. ↑ a b Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, Rothballer A, Ascano M Jr, Jungkamp AC, Munschauer M, Ulrich A, Wardle GS, Dewell S, Zavolan M, Tuschl T .: Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. . In: Cell . 141, No. 1, 2010, pp. 129-141. doi : 10.1016 / j.cell.2010.03.009 . PMID 20371350 . PMC 2861495 (free full text).
  2. ↑ M. Hafner, M. Landthaler, L. Burger, M. Khorshid, J. Hausser, P. Berninger, A. Rothballer, M. Ascano, AC Jungkamp, ​​M. Munschauer, A. Ulrich, GS Wardle, S. Dewell , M. Zavolan, T. Tuschl: PAR-CliP - a method to identify transcriptome-wide the binding sites of RNA binding proteins. In: J Vis Exp. (2010), Volume 41. doi: pii: 2034, 10.3791 / 2034. PMID 20644507 ; PMC 3156069 (free full text).