Immunoprecipitation of the RBP of interest is followed by the isolation of the cross-linked and co-immunoprecipitated RNA. Irradiation of the cells by UV light of 365 nm induces efficient cross-linking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. PAR-CLIP (photoactivatable ribonucleoside–enhanced cross-linking and immunoprecipitation) is a biochemical method used for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs).
#Iclip technique free#
The RNA-protein complexes are then separated from free RNA using gel electrophoresis and membrane transfer. In order to allow for sequence specific priming of reverse transcription, RNA adapters are ligated to the 3' ends, while radiolabeled phosphates are transferred to the 5' ends of the RNA fragments. The cross-linked cells are then lysed, and the protein of interest is isolated via immunoprecipitation.
![iclip technique iclip technique](https://epigenie.com/wp-content/uploads/2013/12/azaip.png)
Upon UV exposure, covalent bonds are formed between proteins and nucleic acids that are in close proximity. CLIP begins with the in-vivo cross-linking of RNA-protein complexes using ultraviolet light (UV).