RIP protocol PDF

Interest in RNA-protein interactions is booming as we begin to appreciate the role of RNA, not just in well-established processes such as transcription, splicing, and translation, but also in newer fields such as RNA interference and gene regulation by non-coding RNAs. RIP is an antibody-based technique used to map RNA–protein interactions in vivo by immunoprecipitating the RNA binding protein of interest together with its associated RNA and allows identification of bound transcripts. RIP precipitates a specific RNA binding protein (RBP) and associated RNA (mRNAs, non coding RNAs, viral RNAs) that can be detected by real- time PCR, microarrays or e.g. sequencing. Here is a RIP protocol adapted from Khalila et al. PNAS 2009, Hendrickson et al. 2009, Hendrickson et al. 2008 and from Rinn et al. Cell 2007.

Reagents

Nuclear isolation buffer

1.28 M sucrose
40 mM Tris-HCl pH 7.5
20 mM MgCl₂
4% Triton X-100

RIP buffer

150 mM KCl
25 mM Tris pH 7.4
5 mM EDTA
0.5 mM DTT
0.5% NP40
100 U/ml RNAase inhibitor SUPERASin (add fresh each time) 
Protease inhibitors (add fresh each time)

RIP protocol:

1. Cell Harvesting (optional treatment of cells with formaldehyde to cross-link in vivo Protein-RNA complexes)
2. Nuclei isolation and nuclear pellets lysis
3. Shearing of chromatin
4. Immunoprecipitation to purify the RNA binding protein (RBP) of interest together with the bound RNA
5. Washing off unbound material
6. Purification of RNA that is bound to immunoprecipitated RBP
7. Reverse transcription (RT) of RNA to cDNA and analysis (qPCR of cDNA if target is known; if target is not known create cDNA libraries, microarrays and sequencing can be used for analysis)

1. Cell Harvesting

1. Grow cells of the tissue culture cell line of interest to confluency and treat cells as required for the experiment.
2. If a cross-linking step is required this will require optimization of the fixation time, check out the Cross-linking section of ourChIP protocol for details.
3. Harvest cells by trypsinization and resuspended in PBS (e.g. 10x7 cells in 2 ml PBS), freshly prepared nuclear isolation buffer (2 ml) and water (6 ml), keep on ice for 20 min (with frequent mixing).

4. *One or more negative controls should be maintained throughout the experiment, e.g. no-antibody sample or immunoprecipitation from knockout cells or tissue, knockdown cells are not recommended for negative control experiments

2. Nuclei isolation and nuclear pellets lysis

1. Pellet nuclei by centrifugation at 2,500 g for 15 min.
2. Resuspend nuclear pellet in freshly prepared RIP buffer (1 ml).

3. *Avoid contamination using RNase-free reagents such as RNase-free tips, tubes and reagent bottles; also use ultraPURE distilled, DNase-free, RNase-free water to prepare buffers and solutions.

3. Shearing of chromatin

1. Split resuspended nuclei into two fractions of 500 ml each (for Mock and IP).
2. Mechanically shearing using a dounce homogenizer with 15–20 strokes.

3. *Different cell lines might require optimization of shearing conditions.

4. Pellet nuclear membrane and debris by centrifugation at 13,000 rpm for 10 min.
5. *Freeze an aliquot of lysate in liquid nitrogen for reference RNA isolation. *Stringent washing of protein A/G bead pellets is important and might need to be optimised.

4. RNA Immunoprecipitation

1. Add antibody to protein of interest (2 to 10 ug) to supernatant (6 mg-10 mg) and incubate for 2 hr (to overnight) at 4^oC with gentle rotation.
2. Add protein A/G beads (40 µl) and incubate for 1 hr at 4^oC with gentle rotation.

3. *The amount of antibody that is added and the incubation time might need to be optimised depending on the protein of interest and antibody. If an antibody is working in IP, this is a good indication that it will work in RIP.

5. Washing off unbound material

1. Pellet beads at 2,500 rpm for 30 s, remove supernatant, and resuspend beads in 500 ml RIP buffer.

2.  

3. Repeat for a total of three RIP washes, followed by one wash in PBS.

4. *Freeze five percent of the beads for SDS PAGE analysis after the second wash (e.g. use 5 μl of bead slurry 
   if you have 100 μl total bead slurry volume)

6. Purification of RNA that was bound to immunoprecipitated RBP

1. Isolate coprecipitated RNAs by resuspending beads in TRIzol RNA extraction reagent (1 ml) according to manufacturer’s instructions (further information can be found in our RNA isolation protocol).
2. Elute RNA with nuclease-free water (e.g. 20 μl).

3. *Add approximately 15-25 μl (depending on yield) of either DEPC treated TE buffer or water to the RNA pellet. 
   Eluted RNA can be stored at -80°C.

4. Protein isolated by the beads can be detected by western blot analysis (further information can be found in our Western blot protocol).

If a cross-linking step has been used (1.2), the cross-link should now be reversed. Check out the reverse cross-links section of ourChIP protocol for details.

7. Reverse transcription (RT) of RNA to cDNA and analysis

1. Reverse transcription of DNAse treated RNA according to manufacturer’s instructions (further information on DNAse treatment and Reverse transcription can be found in our RNA isolation protocol).
2. If target is known use qPCR of cDNA; if target is not known create cDNA libraries, microarrays and sequencing can be used for analysis.

3. *The control experiments should give no detectable products after PCR amplification, and high-throughput sequencing of these control libraries should return very few unique sequences.

Useful references:

Further information on the RIP protocol can be found at:

A. M. Khalila, M. Guttmana, M. Huarte, M. Garbera, A. Rajd, D. R. Morales, K. Thomas, A. Pressera, B. E. Bernstein, A. v. Oudenaardend, A. Regeva, E. S. Lander, and J. L. Rinn, “Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.” PNAS July 14 2009.

D. G. Hendrickson, D. J. Hogan, H. L. McCullough, J. W. Myers, D. Herschlag, J. E. Ferrell, and P. O. Brown, “Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human microRNA.” PLoS Biology 2009.

D. G. Hendrickson, D. J. Hogan, D. Herschlag, J. E. Ferrell, and P. O. Brown, “Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance.” PLoS One 2008.

J. L. Rinn, M. Kertesz, J. K. Wang, S. L. Squazzo, X. Xu, S. A. Brugmann, L. H. Goodnough, J. A. Helms, P. J. Farnham, E. Segal, and H. Y. Chang “Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.” Cell 129:1311–1323, 2007.

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