RNA Immunoprecipitation (RIP) Protocol
Licia Miller Product Manager
RNA immunoprecipitation (RIP) technology is an experimental method specifically used to study the interaction between RNA and proteins in cells. It is used to map RNA-protein interactions and RNA modifications (such as m6A and ac4C) in vivo. This technology is based on the specific binding of RNA to protein. By using specific antibodies to enrich RNA molecules containing RNA binding proteins (RBPs) with target RNAs, the interaction relationship between RNA and proteins is revealed, thereby understanding the regulatory mechanism of specific RNAs or the biological processes involved.
RIP technology allows researchers to explore the dynamic process of post-transcriptional regulatory networks and helps to discover regulatory targets of miRNAs. In this way, nucleic acids associated with specific RNA binding proteins can be identified, including mRNA, non-coding RNA (such as long non-coding RNA, enhancer RNA, miRNA) and viral RNA.
Phase 1 Cell harvesting and nuclei isolation
1. Grow cells to confluence and optimize fixation time if a cross-linking step is required.
2. Aspirate the culture medium and wash the cells twice with ice-cold PBS to remove the culture medium and other impurities.
3. Harvest cells by trypsinization and resuspend in PBS (e.g., 2 mL PBS for 107 cells), freshly prepared nuclei isolation buffer (2 mL) , and water (6 mL) . Place on ice for 20 minutes (stir occasionally).
4. Centrifuge at 2,500 g for 15 minutes at 4°C to pellet the nuclei.
5. Resuspend the nuclear pellet in 1 mL of freshly prepared RIP buffer. Use RNase- free reagents (e.g., RNase- free pipette tips, tubes, and reagent bottles) to avoid contamination; also use ultrapure distilled water, DNase-free, and RNase-free water to prepare buffers and solutions.
6. (Optional) Take 10 µl of protein lysate and label it "Input". Store it in a -80℃ refrigerator until you take it out in step 5 and perform RNA purification experiment simultaneously with IP sample.
Phase 2 Chromatin shearing
1. Divide the suspended nuclei into two parts, 500 µL each, one for mock experiment and the other for IP.
2. Mechanically shear the chromatin using a homogenizer at 15-20 strokes.
3. Centrifuge at 13,000-16,000 × g for 10 minutes to pellet nuclear membrane and debris. Freeze an aliquot of the lysate in liquid nitrogen as a control for RNA isolation.
Phase 3 RNA immunoprecipitation
1. Add 2-10 ug of the antibody of the target protein to the supernatant (6-10 mg) and incubate at 4°C with gentle rotation for 2 hours or overnight.
2. Add 40 µL of Protein A/G magnetic beads and incubate at 4°C with gentle rotation for 1 hour.
Stage 4 Washing
1. Centrifuge at 2,500 rpm for 30 s to pellet the beads, remove the supernatant, and resuspend the beads in 500 µL RIP buffer.
2. Repeat the wash step three times with RIP buffer and once with PBS. After the second wash, freeze 5% of the beads for SDS-PAGE analysis (e.g., if the total slurry volume is 100 µL, use 5 µL of slurry).
Stage 5 Purification
1. Add 1 mL of TRIzol RNA Extraction Reagent to resuspend the magnetic beads to isolate the co-precipitated RNA.
2. Elute the RNA with 20 µL (15 - 25 µL depending on the yield) of DEPC-treated TE buffer or nuclease-free water. The eluted RNA can be stored at -80°C.
3. Proteins separated by magnetic beads can be detected by Western blot analysis. Please refer to our Western blot protocol.
4. If a cross-linking step has been used, the cross-linking should now be reversed.
Phase 6 Results Analysis
1. Use DNA reverse transcriptase to reverse transcribe the purified RNA into cDNA.
2. If the target is known , it can be analyzed directly by qPCR of cDNA. If the target is unknown, it can be analyzed by establishing a cDNA library, microarray and sequencing.
Control experiments should yield no detectable products after PCR amplification, and high-throughput sequencing of these control libraries should return very few unique sequences.
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