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Isolation and detection of RNA by co-immunoprecipitation
The discovery of non-coding RNA has reinvigorated the RNA field as a central focus in life science research. As an inherently unstable biological macromolecule, most RNA molecules require interaction with specific RNA-binding proteins to form stable RNA/protein complexes within cells. These dynamic interactions play a crucial role throughout the entire RNA life cycle—from transcription and processing to transport, localization, function, and degradation. Therefore, isolating or identifying RNA-binding proteins is a key technique for uncovering functional RNA molecules. This typically involves immunoprecipitating RNA-protein complexes using antibodies against RNA-binding proteins, followed by RNA isolation from the complex. The isolated RNA can then be analyzed using end labeling and denaturing gel electrophoresis, or through high-throughput RNA sequencing for comprehensive sequence profiling.
All solution reagents used were either DEPC-treated or prepared in DEPC water to minimize RNase contamination. During RNA extraction, the rinsing step may be omitted if necessary. For RNA sample dissolution and storage, TE buffer (pH 8.0) is recommended. If subsequent enzymatic reactions are planned, store at 80°C; for electrophoresis, use 100% deionized formamide. Skilled handling of RNA is essential, and using an RNAZap environment helps reduce RNase contamination.
**Protocol Steps:**
1. **Immunoprecipitation of RNP complexes from tissue or cell lysate:**
- Pre-UV cross-link RNA-binding protein complexes in tissues or cells.
- Wash tissue or cells twice with ice-cold PBS.
- Homogenize tissue samples in lysis buffer (25 mmol/L Tris-HCl pH 7.5, 150 mmol/L KCl, 2 mmol/L EDTA, 0.5% NP40, 1 mmol/L NaF, 1 mmol/L DTT, 100 U/ml RNasin, and EDTA-free protease inhibitors). Cell homogenization may be skipped.
- Centrifuge at 14,000 rpm (16,000 g) for 10 minutes at 4°C. Use the supernatant for immunoprecipitation.
- Incubate Protein A/G Sepharose beads with antibody at 4°C for about 6 hours.
- Add the supernatant to the bead-antibody mixture and incubate for 6–12 hours or overnight.
- Centrifuge and wash the beads with low-salt and high-salt buffers to remove non-specific binding.
2. **Extraction of small RNA:**
- Resuspend beads in Proteinase K solution and digest at 55°C for 10 minutes.
- Extract RNA using the TRizol method and precipitate with ethanol.
3. **RNA 5' end labeling and electrophoresis detection:**
- Treat RNA with Calf Intestinal Phosphatase to remove 5' phosphate groups.
- Label the 5' ends with γ-32P ATP after phenol-chloroform extraction and ethanol precipitation.
- Separate labeled RNA on a 15% polyacrylamide urea denaturing gel and visualize via autoradiography.
4. **Solexa High-throughput RNA Sequencing:**
- Prepare RNA libraries for next-generation sequencing to identify and quantify RNA species across the transcriptome.