iCLIP

ICLIP ( engl. individual- nucleotide resolution cross- linking and immunoprecipitation, cross-linking and immunoprecipitation with single nucleotide resolution ' ) is a method for determining the biochemistry of RNA - protein interactions. Such interactions take place eg ribonucleoproteins and protein complexes containing micro- RNA.

Principle

The iCLIP uses a combination of different methods. After cross-linking of the RNA-protein complex in cell cultures to UV - light, the bound molecules can be isolated together with existing antibodies in the course of an immunoprecipitation after cell disruption together due to the selective binding of the protein portion. RNA-protein crosslink the molecules are subjected to the subsequent detection by means of a radioactivity of a marker of the RNA component with radioactive phosphorus. This is followed by protein purification by SDS- PAGE with detection by Western blot. The complexes are extracted from the blot membrane to release by a proteinase K digestion of the protein fractions of the crosslinked complexes previously cross-linked RNA. The RNA is purified by phenol-chloroform extraction, and used in a RT-PCR for the production of cDNA with the same sequence of nucleobases. CDNA is purified in a DNA -polyacrylamide gel electrophoresis followed by gel extraction and ethanol precipitation. The purified DNA is sequenced by DNA sequencing in high throughput. As the determined sequences of the cDNA corresponding to that of the RNA, thus the sequence of all the RNA, it is determined on the basis of the previously mitaufgereinigt specific binding to a particular protein.

ICLIP has similarities with the CLIP -Seq, the PAR- CLIP, however, is considerably more complex with 64 reaction steps. In comparison, however, it can prove directly the cross-linked nucleotide. By SDS -PAGE, an additional protein purification, increasing the amount of kontaminanter and unwanted RNA in RT -PCR decreases, leading to the identification of false positive sequences.