RNA Immunoprecipitation (RIP, RIP-chip, RIP-seq)

RIP can be coupled to microarray (RIP-chip) or sequencing (RIP-seq) to identify RNAs that are bound by an RBP of interest. Both RIP-chip and RIP-seq rely on the specificity or RBP-RNA interaction in cell extract to preserve the interaction long enough to isolate via immunoprecipitation.

RIP-chip was first described in 2004 (Keene et al., 2004). After next-gen sequencing was established, RIP-seq emerged, similar to development of ChIP-seq from ChIP-chip. RIP-seq was first used in 2008 (Cloonan et al., 2008). The major advantage of RIP is that it is requires little specialized equipment or reagents. The disadvantages compared to CLIP include lack of actually RBP binding site identification, non-specific RNA interaction identification, and high signal-to-noise ratio (Mili and Steitz, 2004).

A noteworthy special application of RIP-chip is recombinant RIP-chip (rRIP-chip). rRIP-chip uses a recombinant protein of interest to probe an isolated total RNA sample. The recombinant protein then allows easy isolation of target RNAs which are identified by microarray (Townley-Tilson et al., 2006).

RIP Additional Reading

Jain, R., Devine, T., George, A.D., Chittur, S.V., Baroni, T.E., Penalva, L.O., and Tenenbaum, S.A. (2011). RIP-Chip analysis: RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling. Methods Mol. Biol. 703, 247-263.

This is a textbook chapter that describes the RIP protocol, but it also includes a substantial background on RIP technologies that includes some good information and references.

Dahm, G.M., Gubin, M.M., Magee, J.D., Techasintana, P., Calaluce, R., and Atasoy, U. (2012). Method for the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts using RIP-Chip. J. Vis. Exp. (67). pii: 3851. doi, 10.3791/3851.

This paper includes a video that demonstrates the step-by-step protocol for performing RIP-chip.

Reference List

• Cloonan, N., Forrest, A.R., Kolle, G., Gardiner, B.B., Faulkner, G.J., Brown, M.K., Taylor, D.F., Steptoe, A.L., Wani, S., Bethel, G., et al. (2008). Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat. Methods 5, 613-619.
• Keene, J.D., Komisarow, J.M., and Friedersdorf, M.B. (2006). RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat. Protoc. 1, 302-307.
• Mili, S., and Steitz, J.A. (2004). Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA 10, 1692-1694.
• Townley-Tilson, W.H., Pendergrass, S.A., Marzluff, W.F., and Whitfield, M.L. (2006). Genome-wide analysis of mRNAs bound to the histone stem-loop binding protein. RNA 12, 1853-1867.