Table 2.
Variant CLIP protocols
| Name | Description | Advantages | Disadvantages | Reference(s) to method |
|---|---|---|---|---|
| CLIP | The original CLIP protocol was used to cross-link RNA–protein complexes in mouse brain. See Table 1. | Inexpensive; method most fully vetted | Low complexity; requires immunoprecipitating antibody to endogenous protein | Ule et al. 2003, 2005; Jensen and Darnell 2008; http://lab.rockefeller.edu/darnell/methods/CLIP |
| HITS-CLIP | Use of next-generation sequencing to develop genome-wide RNA–protein maps. Originally used in mouse brain. See Table 1. | High complexity; method most fully vetted; able to map RNA–protein cross-link sites by mutagenesis | Licatalosi et al. 2008 | |
| Crac CLIP | Uses affinity tags to immunoprecipitate RNABP | Useful when immunoprecipitating antibody to endogenous protein is not available | Unless tag is knocked into endogenous locus, stoichiometry of RNABP and hence RNA maps may be affected. RNABP-tag fusion construct may affect RNA-binding properties. | Granneman et al. 2009 |
| PAR-CLIP | 4-Thio-uridine incorporation into RNA allows more efficient cross-linking. | Useful if high cross-linking efficiency is needed, or if native proteins are inefficiently cross-linked; able to map RNA–protein interaction sites | Method currently restricted to tissue culture; 4-thio-U is toxic to cells. Analysis scoring T-to-C transitions restricts CLIP sequence space. | Hafner et al. 2010 |
| iCLIP | CLIP variant that maps sites of reverse-transcriptase pausing at or near sites of amino acid–nucleic acid cross-link | Able to map RNA–protein cross-link sites; efficient capture of cross-linked RNA tags | Requires immunoprecipitating antibody to endogenous protein | Konig et al. 2010 |
