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Ribonucleoprotein Immunoprecipitation (RIP) Protocol

Ribonucleoprotein (RNP) is a combination of RNA and RNA-binding proteins that drives the regulation of post-transcriptional gene expression. A detailed understanding of RNPs provides valuable information for not only the knowledge of particular pathways, but also development of novel compounds representing potential therapeutic targets and biomarkers.

RNA immunoprecipitation (RIP) is an antibody-based technique that allows the immunoprecipitation and isolation of transcripts (mRNAs or microRNAs) and protein components of RNP complexes from cell extracts. RNAs can be extracted from the purified RNA-protein complexes and further detected by various molecular biology tools including cDNA sequencing, microarrays or real-time PCR. This protocol provides a detailed procedure to obtain high-qualify RNA for a successful RIP assay.

Reagents:

  • Polysome lysis buffer: 150 mM KCl, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, 0.5% NP-40, 0.5 mM DTT, with 100 U/ml RNAase inhibitor and 1× protease inhibitor cocktail.
  • NT2 buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40, with 1× protease inhibitor cocktail.
  • NT2-Ab coupling buffer: NT2 buffer supplemented with 5% BSA.
  • Nuclease-free PBS: pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4.

Equipments:

  • RNase-free tips, tubes and reagent bottles
  • Vortex
  • Eppendorf tube rotator
  • Refrigerated microcentrifuge

Steps:

Pre-step: Antibody coating of beads

1. Take 75 μL Protien A or G-agarose beads slurry, and wash three times with 500 μL NT2 buffer.

2. Centrifuge at 2,000 × g for a few seconds at 4°C and then discard the supernatant.

3. Resuspend with 100 μL NT2-Ab coupling buffer, add 2~20 μg relevant antibodies of interest or normal IgG.

4. Vortex, and incubate at 4 °C overnight with gentle rotation on a rotating device.

5. Centrifuge at 2,000 × g for a few seconds at 4°C, and aspirate the supernatant.

6. Resuspend with 1 mL NT-2 buffer, mix and centrifuge at 2,000 × g for a few seconds at 4°C.

7. Aspirate the supernatant, and repeat Step 5-6 for additional five times.

8. Resuspend the beads with 850 μL NT-2 buffer (with 100 U/ml RNAase inhibitor), and keep the tube on ice.

Cross-link (optional) and cell harvest

9. Freshly cultured cells are suggested in this protocol to obtain high-quality RNAs. If a cross-linking step is required, follow the cross-link procedure of ChIP protocol.

10. Collect 0.5 - 1 × 107 cells into a 15-mL conical tube, centrifuge at 2,000 × g at 4°C for 5 min, and discard the supernatant.

11. Wash the cell pallet with ice-cold PBS for twice, centrifuge at 2,000 × g at 4°C for 5 min, and discard the supernatant.

Prepare RNP lysates

12. Add 1 mL cold polysome lysis buffer, and vortex vigorously.

13. Incubate the tube on ice for 10 minutes.

14. Centrifuge at 14,000 × g at 4°C for 10 min, and retain the supernatant.

15. Take 10% of the lysate as input for the analysis of protein (western blot) or total RNA, freeze at -80 °C until further use.

RNP Immunoprecipitation

16. Add 150 μL lysate into beads slurry (obtained in Step 8), mix and incubate at 4°C overnight with gentle rotation.

17. Centrifuge at 12,000 × g for 1 min, and discard the supernatant.

18. Wash six times with 1 mL cold NT-2 buffer to remove unbound material.

RNA purification and analysis

19. Add Trizol reagent directly to the beads, and isolate the RNA from the immunoprecipitated pellet according to the manufacturer’s instructions.

20. Dissolve purified RNA in nuclease-free water, and can be stored at -80 °C.

21. Use 2~10 μL of the RNA sample to prepare cDNA for further analysis.

Learn about the principle of RIP

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References:

1. Hassan MQ, et al., Ribonucleoprotein immunoprecipitation (RNP-IP): a direct in vivo analysis of microRNA-targets., J Cell Biochem., 2010,1;110(4):817-22.
2. Jayaseelan S, et al., RIP: an mRNA localization technique., Methods Mol Biol.,2011, 714:407-22.
3. Liu, F. RNP-IP (Original protocol)-getting majority of RNA from RNA binding protein in nucleus. Bio-protocol., 2011, Bio101: e218.

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