RIP is an antibody-based technique used to map in vivo RNA-protein interactions. The RNA binding protein (RBP) of interest is immunoprecipitated together with its associated RNA for identification of bound transcripts (mRNAs, non-coding RNAs or viral RNAs). Transcripts are detected by real-time PCR, microarrays or sequencing.
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.
The following RIP protocol is adapted from Khalilaet al. (2009), Hendrickson et al. (2009), Hendrickson et al. (2008) and Rinnet al. (2007).
Grow cells to confluency and treat as required for the experiment.
Harvest cells by trypsinization and resuspend in PBS (e.g. 107 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).
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.
Nuclei isolation and lysis of nuclear pellets
Pellet nuclei by centrifugation at 2,500 g for 15 min.
Resuspend nuclear pellet in freshly prepared RIP buffer (1 mL).
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.
Split resuspended nuclei into two fractions of 500 mL each (for mock and IP).
Mechanically shear chromatin using a dounce homogenizer with 15–20 strokes. Different cell lines might require optimization of shearing conditions.
Pellet nuclear membrane and debris by centrifugation at 13,000 rpm for 10 min.
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 optimized.
Add antibody to protein of interest (2–10 mg) to supernatant (6–10 mg) and incubate for 2 h (to overnight) at 4°C with gentle rotation.
Add protein A/G beads (40 µL) and incubate for 1 h at 4°C with gentle rotation.
The amount of antibody that is added and the incubation time might need to be optimized 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.
Washing off unbound material
Pellet beads at 2,500 rpm for 30 s, remove supernatant, and resuspend beads in 500 mL RIP buffer.
Repeat for a total of three RIP washes, followed by one wash in PBS.
Freeze 5% 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).
Purification of RNA that was bound to immunoprecipitated RBP
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).
Elute RNA with nuclease-free water (e.g. 20 μL).
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.
Protein isolated by the beads can be detected by western blot analysis (further information can be found in our western blot protocol).
Reverse transcription (RT) of RNA to cDNA and analysis
Reverse transcribe DNAse treated RNA according to manufacturer’s instructions (further information on DNAse treatment and reverse transcription can be found in our RNA isolation protocol.
Analyze by qPRC of cDNA if the target is known. If target is not known, creation of cDNA libraries, microarrays and sequencing can be used for analysis. 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.
Nuclear isolation buffer
1.28 M sucrose
40 mM Tris-HCl pH 7.5
20 mM MgCl2
4% Triton X-100
150 mM KCl
25 mM Tris pH 7.4
5 mM EDTA
0.5 mM DTT
100 U/ml RNAase inhibitor SUPERase•in™ (add fresh each time)