RIP technology

Summary

Studying RNA-protein binding in cells

Principle

1. Capture of endogenous RNA-binding proteins in the nucleus or in the cytoplasm with antibodies or epitope markers.

2. preventing the binding of non-specific RNA.

3. immunoprecipitation to isolate RNA-binding proteins together with their bound RNA.

4. Bound RNA sequences are identified by microarray (RIP-Chip), quantitative RT-PCR or high-throughput sequencing (RIP-Seq) methods.

Appliance

cell research

Operation method

RIP technology

Materials and Instruments

Material】cells, antibodies, magnetic beads
【Reagents】Cold PBS, RIP, RNase-free ethanol, chloroform, isoamyl alcohol, Salt SolutionⅠ, Salt SolutionⅡ, RIP Wash Buffer, Proteinase K, 10% SDS, Precipitate EnhancerRIP Wash Buffer R, IP Immunoprecipitation Buffer, RIP Wash Buffer, 0.5M EDTA, RNase Inhibitor.
Instruments and Consumables] Petri dishes, enpendoff tubes, centrifuges, magnetic racks, vortex shakers, ice boxes, guns, gun heads, ultra-clean table, 360° rotator

Move

(i). Cell lysate acquisitionA. Monolayer or adherent cell processing1. Wash the cells in the petri dish or culture flask twice with cold PBS.2. Add cold PBS and scrape the cells with a cell scraper and collect them into enpendoff tubes.3. centrifuge the cells at 1500rpm for 5min at 4℃, discard the supernatant and collect the cells.4. Resuspend the cells with a volume of RIP lysate equal to that of the cells, blow well and leave on ice for 5min. 5.5. Dispense 200ul of cell lysate into each tube and store at -80℃.B. Suspension cell processing: first collect cells and then count, then wash and lysedC. Tissue sample processing1. Wash freshly cut tissue three times with cold PBS2. After adding cold PBS, use a homogenizer or other cell separation equipment to disperse the tissue into individual cells and count them3. centrifuge at 1500rpm, 4℃ for 5min, discard the supernatant, and collect the cells.4. Resuspend the cells with a volume of RIP lysate equal to that of the cells, blow well and place on ice for 5 min.5. Dispense 200ul of cell lysate into each tube and store at -80℃.(ii). Preparation of magnetic beadsA. Preparation before experiment1, enpendoff tube2. Magnetic rack3, ice box, RIP Wash Buffer on ice4. Antibody on ice5、Vortex shaker6, gun, gun head in the ultra-clean table irradiation 30min, gun spray DEPC waterB. Preparation of magnetic beads1. Resuspend the magnetic beads2. Label the enpendoff tubes for the experiment, the samples include the target sample, negative control and positive control.3. Pipette 50ul of the resuspended magnetic bead suspension into each enpendoff tube.4. Add 500ul of RIP Wash Buffer to each tube and vortex.5. Place the enpendoff tube on a magnetic rack and rotate it 15° from side to side so that the magnetic beads adhere to a straight line, remove the supernatant and repeat.6. Resuspend the beads with 100ul of RIP Wash Buffer and add about 5ug of the appropriate antibody to each sample.7. Incubate at room temperature for 30min8. Place the enpendoff tube on a magnetic rack and discard the supernatant.9. Add 500ul of RIP Wash Buffer, vortex and shake, discard supernatant and repeat.10. Add 500ul RIP Wash Buffer, vortex and shake, place on ice.(iii). RNA binding protein immunoprecipitationA. Preparation1. Ice box2. 360° rotator3, RIP Wash Buffer, 0.5M EDTA, RNase Inhibitor on ice.B. RNA binding protein immunoprecipitation procedure1. Prepare RIP Immunoprecipitation Buffer.2. Place the enpendoff tubes from the previous step on a magnetic rack, remove the supernatant and add 900ul of RIP Immunoprecipitation Buffer to each tube. 3.3. rapidly thaw the cell lysate prepared in the first step, centrifuge at 14,000rpm, 4℃ for 10min, aspirate 100ul of supernatant into the magnetic bead-antibody complex of the previous step, so that the total volume is 1ml.4. Incubate at 4°C for 3h to overnight5. Centrifuge briefly, place the enpendoff tube on a magnetic rack and discard the supernatant.6. Add 500ul of RIP Wash Buffer, vortex and shake, place the enpendoff tube on a magnetic rack, discard the supernatant and repeat the wash 6 times.(iv). RNA purificationA. Pre-laboratory preparation1. gun, gun head, enpendoff tube UV irradiation for 30 min, spray DEPC water to remove RNA enzyme2. Salt SolutionⅠ, Salt SolutionⅡ, RIP Wash Buffer, Proteinase K, 10% SDS, Precipitate Enhancer on ice.3. RNase-free ethanol, chloroform, and isoamyl alcohol.4. DEPC water on ice5. Centrifuge pre-cooling6. Ice boxB. RNA purification process1. Prepare Proteinase K Buffer. 150ul for each sample.2. Resuspend the above bead-antibody complexes with 150ul of Proteinase K Buffer.3. Incubate at 55℃ for 30min4. After incubation, place the enpendoff tubes on a magnetic rack and draw the supernatant into a new enpendoff tube.5. Add 250ul of RIP Wash Buffer to each tube of supernatant.6. Add 400ul of phenol:chloroform:isoamyl alcohol to each tube, vortex for 15s, and centrifuge at 14,000rpm for 10min at room temperature.7. Carefully aspirate 350ul of the upper aqueous phase into a new enpendoff tube.8. Add 400ul of chloroform to each tube, vortex for 15s, and centrifuge at 14,000rpm for 10min at room temperature.9. Carefully aspirate 300ul of the upper aqueous phase into a new enpendoff tube.10. Add 50ul of Salt SolutionⅠ, 15ul of Salt SolutionⅡ, 5ul of Precipitate Enhancer and 850ul of anhydrous ethanol (without RNase) to each tube, mix, and keep at -80℃ for 1h until overnight.11. Centrifuge at 14,000rpm, 4℃ for 30min, carefully remove the supernatant. 12.12. Rinse once with 80% ethanol, centrifuge at 14,000rpm, 4℃ for 15min, remove supernatant carefully and air dry.13. Dissolve 10-20ul DEPC in water, store at -80℃, send for sequencing or downstream experiments.

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