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Immunoprecipitation

TriLink offers a 3' iCLIP Primer for use iCLIP applications. UV-Crosslinking immunoprecipitation (CLIP) is a powerful technique for elucidating RNA-protein interactions. Standard protocols often employ labeled adapters to visualize the protein-RNA complex. The method involves irreversible crosslinking of the RNA to protein, immunoprecipitation, ligation of 5′ and 3′ adapters, reverse transcription, and finally PCR amplification. Unfortunately, reverse-transcriptase cannot read through crosslinked areas, and this creates a truncated cDNA without the 3′ and 5′ adapters needed to initiate PCR amplification.

A variation of the CLIP method, individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) circumvents this problem through the use of a preadenylated primer (iCLIP primer) and circularization of the reverse transcription product. This method also resolves the binding site to individual nucleotides and increases accuracy for binding sites located within repetitive motifs.

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