CLIP-rtPCR (site-specific analysis)

CLIP-rtPCR is sometimes used to demonstrate RBP interaction with a specific RNA or a site on RNA. Since iCLIP analysis is based on the frequent RT stalling at crosslinking sites, how does that affect the design and interpretation of CLIP-rtPCR? Presumably both forward and reverse PCR primers should be 3’ to crosslinking sites, so that cDNAs can be efficiently produced from the purified RNA fragments? How frequent is the stalling?

Stalling (i.e., cDNA truncation) depends on many factors, but especially the RT conditions (see https://pubmed.ncbi.nlm.nih.gov/28790018/). Various studies tend to estimate it between 3-30% depending on RBP, RT condition, dataset, so quite a wide range. So CLIP-rtPCR may work to some extent even if primers are not just downstream of the crosslinking region. That said, there is also another issue - if applications of CLIP-rtPCR don’t use the SDS-PAGE purification step (which helps remove the non-crosslinked RNA), it’s possible that non-crosslinked RNA will be present, which could be overrepresented in the readout (because there’s no signal loss due to crosslink-induced truncation). It’s also likely that the protein crosslinks to many sites on the RNA, so RNA fragments will exist where crosslink doesn’t overlap with the amplified region (especially if low RNAse is used, so that RNA fragments are long).