iCLIP

iCLIP[1] (individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation) is a method used for identifying protein-RNA interactions. The method uses UV light to covalently bind proteins and RNA molecules. This cross-linking step has generally less background than RNA immunoprecipitation protocols.[2][3] As with all CLIP methods, iCLIP allows for a very stringent purification of the linked protein-RNA complexes, using immunoprecipitation followed by SDS-PAGE and transfer to nitrocellulose. The radiolabelled protein-RNA complexes are then excised from nitrocellulose, and treated with proteinase to release the RNA, leaving one or two amino acids at the crosslink site of the RNA.

The RNA is then reverse transcribed, causing cDNAs to often truncate at the crosslink site, which is the key insight and unique feature in the development of iCLIP, as it allows identification of the site of RNA-protein interaction at high resolution. Analogous resolution can be obtained with standard HITS-CLIP methods, using the observation that RT that does read through the cross link site has a high error rate, which can be used to deduce the position of the crosslink ("Crosslink induced mutation site" (CIMS) analysis[4]).

The small amount of resulting cDNAs is then PCR amplified and sequenced using a next-generation sequencing platform. The primer used for reverse transcription can contain a random sequence, which can be used to barcode cDNAs. This helps to identify PCR over-amplification effects in the high-throughput sequencing step and therefore improves the quantification of binding events.

References
  1. König, Julian; Zarnack, Kathi; Luscombe, Nicholas M.; Ule, Jernej (18 January 2012). "Protein–RNA interactions: new genomic technologies and perspectives". Nature Reviews Genetics. 13 (2): 77–83. doi:10.1038/nrg3141. PMID 22251872.
  2. Yao, Chengguo; Weng, Lingjie; Shi, Yongsheng (20 February 2014). "Global protein-RNA interaction mapping at single nucleotide resolution by iCLIP-seq". Methods in Molecular Biology. 1126: 399–410. doi:10.1007/978-1-62703-980-2_27. PMC 4412157. PMID 24549678.
  3. Huppertz, Ina; Attig, Jan; D'Ambrogio, Andrea; Easton, Laura E; Sibley, Christopher R; Sugimoto, Yoichiro; Tajnik, Mojca; König, Julian; Ule, Jernej (February 2014). "iCLIP: protein-RNA interactions at nucleotide resolution". Methods. 65 (3): 274–87. doi:10.1016/j.ymeth.2013.10.011. PMC 3988997. PMID 24184352.
  4. Zhang, Chaolin; Darnell, Robert B (1 June 2011). "Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data". Nature Biotechnology. 29 (7): 607–614. doi:10.1038/nbt.1873. PMC 3400429. PMID 21633356.
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