The discovery of non-coding RNA makes RNA become the focus of life science research once again. Because RNA is an unstable biological macromolecule, most RNAs need to be bound to a specific RNA-binding protein to form an RNA / protein complex in order to be stable in cells. Furthermore, dynamic association between RNA and RNA-binding proteins throughout and accompanies the entire life cycle of RNA transcriptional synthesis, processing and modification, intracellular trafficking and localization, functional development and degradation. Therefore, take advantage of RNA-binding proteins to isolate or discover functional RNA molecules is an indispensable research method in the field of RNA research.
RNA binding protein immunoprecipitation (RIP) assay utilizes antibodies against the target protein to precipitate the corresponding RNA-protein complexes. The RNA bound to the complex can be validated by q-PCR or sequencing analysis after isolation and purification. RIP is a technique to study the combination of RNA and protein in cells. It is a powerful tool to understand the dynamic process of post-transcriptional regulation of the network, which can help us discover the regulatory targets of miRNAs.
Preparation of mRNP lysate
1. Collect enough tissue culture cells to generate 2-5 mg of total protein per RIP. Typically, this is comparable to 5-20×106 mammalian cells. Pellet by centrifugation (~1,000g) for 10 min at 4℃, washing several times with 10 ml of ice cold phosphate-buffered saline (PBS) in a conical tube. Alternatively, whole tissue may be ground using a mechanical homogenizer. Additionally, individual cells derived by micro-dissection may be used. The total amount of protein used per RIP must be optimized based upon the abundance of the RNA-binding protein being investigated as well as the planned method of RNA detection.
2. Resuspend final cell pellet with an approximately equal volume of polysome lysis buffer supplemented with RNase inhibitors and protease inhibitors. Clumps of cells should be broken up by pipetting up and down several times. Allow mRNP lysate to incubate on ice for 5 min and store at -80℃. Lysate may be stored for several months at -80℃. The lysis of certain cell types can be enhanced by pumping the lysate through a small gauge syringe needle.
Note: Immediate freezing of the lysate is essential to complete the lysis process as well as preventing adventitious binding. Additional freeze-thaw cycles should be avoided prevent protein and RNA degradation.
Antibody coating of protein A/G beads
3. At 4℃, pre-swell protein-A Sepharose beads in NT2 supplemented with 5% BSA to a final ratio of 1:5 for at least 1 h before use. Protein G or A/G Sepharose beads may be substituted depending upon the isotype of the antibody being used. Pre-swollen beads may be stored for several months at 4℃ when supplemented with 0.02% sodium azide.
4. In a 1.5 ml microcentrifuge tube, add 250-500 μl of protein A-BSA slurry. After a pulse centrifugation this should yield a pelleted bead volume of ~50 μl at the bottom of the microcentrifuge tube.
5. Add antibody to bead slurry and incubate for 2-18 hours, tumbling end over end at 4℃. The volume of antibody added to the beads is dependent upon antibody titer, but this amount should be more than enough to pull down all available protein being investigated. This mixture may be stored for several weeks at 4℃ when supplemented with 0.02% sodium azide.
Note: In parallel, a control antibody must be used to assess background RNA levels. Typically, this is an isotype-matched antibody or whole normal sera from a matched species. The amount of control antibody should be equal to the amount of immunoprecipitating antibody being used.
6. Immediately before use, wash antibody-coated beads with 1 ml of ice-cold NT2 buffer 4-5 times. To wash, spin down beads by pulsing in an ultracentrifuge at 4℃, remove liquid with hand pipettor or aspirator and resuspend in ice-cold NT2 buffer by flicking the tube several times with a finger. This washing removes unbound antibody as well as contaminants such as RNases, which may be present in the antibody mixture, and is one of the reasons we pre-bind the antibody to beads.
7. After the final wash, resuspend beads in 850 μl of ice-cold NT2 buffer. Add 200 units of an RNase inhibitor (5 μl RNase Out), 2 μl (to final concentration of 400 μM) Vanadyl ribonucleoside complexes, 10 μl of 100 mM DDT and EDTA to 20 mM.
Immunoprecipitation reaction and RNA precipitation
8. Thaw mRNP lysate on ice and centrifuge at 15,000g for 15 min to clear lysate of large particles. Transfer cleared supernatant to microfuge tube and store on ice. Additionally, pre-clearing of lysate with beads may be used to reduce background, if necessary. This may, however, reduce signal.
9. Add 100 μl of cleared lysate to antibody mixture prepared in Step 7.
Note: This dilution of lysate is important to reduce adventitious binding.
10. Immediately flick tube several times with a finger to mix, and centrifuge briefly at 8,000-10,000g to pellet beads. Remove 100 μl of supernatant to represent total cellular mRNA.
11. Incubate for 4 h at 4℃ tumbling end over end. Alternatively, incubate 2 h at room temperature (18-25℃) and times as short as 15 min have worded well in some cases.
12. Pellet beads and save supernatant for later analysis if desired. Supernatant may be stored at -20℃ for several months.
13. Wash beads 4-5 times with 1 ml of ice-cold NT2 buffer by pulsing in an ultracentrifuge and removing supernatant with a hand pipettor or an aspirator.
Note: Thorough washing is critical for reducing background. NT2 buffer may be supplemented with urea, sodium deoxycholate or SDS to increase stringency depending upon the RNA-binding protein being investigated. All tubes should be kept on ice as much as possible while working quickly during the washing process to reduce degradation.
14. Resuspend the beads in 100 μl of NT2 buffer. NT2 buffer can also be supplemented with 30 μg of proteinase K to release the RNP components. Incubate mixture for 30 min at 55℃, flicking the tube occasionally using a finger.
15. Release the RNP components and isolate the RNA form the immunoprecipitated pellet by adding either Trizol reagent or phenol-chloroform-isoamyl alcohol directly to the beads. Precipitate RNA and resuspend in a volume appropriate for subsequent detection method. Addition of glycogen (20 μg) as a carrier to the precipitation reaction aids in making the RNA pellet more readily visible and aids in recovery of RNA.
16. After release of the RNP components, one can isolate the proteins associated with the complex and submit them for standard mass spectroscopy or other proteomics identification procedures. Such information can be very useful in the functional analysis subsequent to the identification of RNA subsets as described above.
Keene, J.D.; et al. RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc. 2006, 1(1):302-307.