8.
Solutions
怎么提升气质RNA Immunoprecipitation (RIP)
RIP is an antibody-bad technique to map RNA–protein interactions in vivo by immunoprecipitating a specific RNA binding protein (RBP) and associated RNA that can be detected by real- time PCR, microarrays quencing.
Nuclear isolation buffer: 1.28 M sucro 40 mM Tris-HCl pH 7.5 20 mM MgCl2 4% Triton X-100
RIP buffer: 150 mM KCl 25 mM Tris pH 7.4 5 mM EDTA 0.5 mM DTT 0.5% NP40 100 U/ml RNAa inhibitor SUPERASin (add fresh each time) Protea inhibitors (add fresh each time)
9. Further information
am职位For a detailed protocol, plea visit /protocols, further information on the RIP protocol can be found at: A. M. Khalila et al., “Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.” PNAS July 14 2009. D. G. Hendrickson,
D. J. Hogan, H. L. McCullough, J. W. Myers, D. Herschlag, J. E. Ferrell, and P . O. Brown, “Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human microRNA.” PLoS Biology 2009. D. G. Hendrickson, D. J. Hogan, D. Herschlag, J. E. Ferrell, and P . O. Brown, “Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance.” PLoS One 2008. J. L. Rinn, M. Kertesz, J. K. Wang, S. L. Squazzo, X. Xu, S. A. Brugmann, L. H. Goodnough, J. A. Helms, P . J. Farnham, E. Segal, and H. Y . Chang “Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.” Cell 129:1311–1323, 2007.
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This leaflet contains a brief summary of the RIP protocol adapted from Khalila et al. 2009, Hendrickson et al. 2009 and 2008, and Rinn et al. 2007.
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RIP Protocol
Tissues/cells, sometimes treated with formaldehyde ( )
1. Cell Harvesting
1.1. Harvest cells by trypsinization, resuspended in PBS (e.g. 10x7 cells in 2 ml), freshly prepared nuclear isolation buffer (2 ml) and water (6 ml), keep on ice (20 min, frequent mixing). Tip: One or more negative controls should be maintained throughout the experiment, e.g. no-antibody sample or immunoprecipitation from knockout cells or tissue.
2. Nuclei isolation and nuclear pellets lysis
2.1. Pellet nuclei by centrifugation (2,500 G, 15 min). 2.2. Resuspend nuclear pellet in freshly prepared RIP buffer (1 ml).
腰细
Cell lysis and chromatin shearing
Tip: Avoid contamination using RNa-free reagents such as RNa-free tips, tubes and reagent bottles; also u ultraPURE distilled, DNa-free, RNa-free water to prepare buffers and solutions.
3. Shearing of chromatin
3.1. Split resuspended nuclei into two fractions of 500 ml each (for Mock and IP). 3.2. U a dounce homogenizer for shearing with 15–20 strokes. 3.3. Pellet nuclear membrane and debris by centrifugation (13,000 rpm, 10 min).
Immunoprecipitation
4. RNA Immunoprecipitation
4.1. Add antibody to protein of interest (2 to 10 μg) to supernatant (6 mg-10 mg), incubate (2 hr to overnight, 4°C, gentle rotation). 4.2. Add protein A/G beads (40 μl), incubate (1 hr, 4°C, gentle rotation). Tip: If an antibody is working in IP , this is a good indication that it will work in RIP .
Washing off unbound material RNA binding protein (RBP)
5. Washing off unbound material
5.1. Pellet beads (2,500 rpm, 30 s), remove supernatant, resuspend beads in RIP buffer (500 ml). 5.2. Repeat for a total of three RIP washes, followed by one wash in PBS. Tip: Optimization and stringent washing conditions are very important.
Bound RNA purification
春雨像什么6. Purification of RNA that was bound to immunoprecipitated RBP
6.1. Isolate coprecipitated RNAs by resuspending beads in TRIzol RNA extraction reagent (1 ml) according to manufacturer’s instructions. 6.2. Elute RNA with nuclea-free water (e.g. 20 μl). 6.3. Protein isolated by the beads can be detected by western blot analysis.
Rever transcribe RNA to cDNA followed by qPCR (if target known) or create cDNA libraries followed by microarrays or quencing (if target unkown)
Figure 1: Schematic reprentation and summary of RIP protocol
7. Rever transcription and analysis
游公园>狼怎么画7.1. Rever transcribe DNA treated RNA according to manufacturer’s instructions. 7.2. If target is known u qPCR of cDNA; if target is not known create cDNA libraries, microarrays and quencing can be ud for analysis. Tip: The control experiments should give no detectable products after PCR amplification, and highthroughput quencing of the control libraries should return very few unique quences.
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