RNA Immunoprecipitation(RIP) protocolRIP实验方法

8.
Solutions
RNA Immunoprecipitation (RIP)
RIP is an antibody-based 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 or e.g. sequencing.
Nuclear isolation buffer: 1.28 M sucrose 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 RNAase inhibitor SUPERASin (add fresh each time) Protease inhibitors (add fresh each time)
9. Further information
For a detailed protocol, please visit https://www.wendangku.net/doc/c82295144.html,/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).
Cell lysis and chromatin shearing
Tip: Avoid contamination using RNase-free reagents such as RNase-free tips, tubes and reagent bottles; also use ultraPURE distilled, DNase-free, RNase-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. Use 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
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 nuclease-free water (e.g. 20 μl). 6.3. Protein isolated by the beads can be detected by western blot analysis.
Reverse transcribe RNA to cDNA followed by qPCR (if target known) or create cDNA libraries followed by microarrays or sequencing (if target unkown)
Figure 1: Schematic representation and summary of RIP protocol
7. Reverse transcription and analysis
7.1. Reverse transcribe DNAse treated RNA according to manufacturer’s instructions. 7.2. If target is known use qPCR of cDNA; if target is not known create cDNA libraries, microarrays and sequencing can be used for analysis. Tip: The control experiments should give no detectable products after PCR amplification, and highthroughput sequencing of these control libraries should return very few unique sequences.
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长链非编码RNA高分综述

Although the central role of RNA in cellular func-tions and organismal evolution has been advocated periodically during the past 50 years, only recently has RNA received a remarkable level of attention from the scientific community. Analyses that compare transcrip-tomes with genomes of mammalian species (BOX 1) have established that approximately two-thirds of genomic DNA is pervasively transcribed, which is in sharp con-trast to the <2% that is ultimately translated into pro-teins 1,2. Moreover, the degree of organismal complexity among species better correlates with the proportion of each genome that is transcribed into non-coding RNAs (ncRNAs) than with the number of protein-coding genes, even when protein diversification by both alterna-tive splicing and post-translational regulation are taken into account 3. This suggests that RNA-based regula-tory mechanisms had a relevant role in the evolution of developmental complexity in eukaryotes. The range of ncRNAs in eukaryotes is vast and exceeds the number of protein-coding genes. Besides the different families of small ncRNAs 4, a large proportion of transcriptomes results in RNA transcripts that are longer than 200 nucleotides, which are often polyadenylated and are devoid of evident open reading frames (ORFs) — these are defined as long ncRNAs (lncRNAs)5–7. Many roles are emerging for lncRNAs in ribonucleo-protein complexes that regulate various stages of gene expression 5,7. Their intrinsic nucleic acid nature confers on lncRNAs the dual ability to function as ligands for proteins (such as those with functional roles in gene regulation processes) and to mediate base-pairing interactions that guide lncRNA-containing complexes to specific RNA or DNA target sites 5,7,8. This dual activ-ity is shared with small ncRNAs 4, such as microRNAs (miRNAs), small nucleolar RNAs and many other small nuclear ribonucleoprotein particles (BOX 2). However, unlike small ncRNAs, lncRNAs can fold into complex secondary and higher order structures to provide greater potential and versatility for both protein and target rec-ognition 5,7,8. Moreover, their flexible 8,9 and modular 10,11 scaffold nature enables lncRNAs to tether protein fac-tors that would not interact or functionally cooperate if they only relied on protein–protein interactions 5,8,12–14. Such combinatorial RNA-mediated tethering activity has enhanced gene regulatory networks to facilitate a wide range of gene expression programmes (FIG. 1) to provide an important evolutionary advantage 5,7,8. This complexity is likely to be further expanded by differential splicing and the use of alternative transcription initiation sites and polyadenylation sites by lncRNAs, thus increasing the number of tethering-module combinations. The expression of lncRNAs has been quantitatively analysed in several tissues and cell types by high-throughput RNA sequencing (RNA-seq) experiments, and it was generally found to be more cell type specific than the expression of protein-coding genes 5,6,8,15–17. Interestingly, in several cases, such tissue specificity has been attributed to the presence of transposable elements that are embedded in the vicinity of lncRNA transcrip-tion start sites 18–20. Moreover, lncRNAs have been shown to be differentially expressed across various stages of differentiation, which indicates that they may be novel ‘fine-tuners’ of cell fate 5–7. This specific spatiotemporal expression can be linked to the establishment of both 1 Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.2 Institute of Molecular Biology and Pathology of the National Research Council, Piazzale Aldo Moro 5, 00185 Rome, Italy.3 Istituto Pasteur Fondazione Cenci Bolognetti, Piazzale Aldo Moro 5, 00185 Rome, Italy.4 Center for Life Nano Science @Sapienza, Istituto Italiano di T ecnologia, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy. Correspondence to I.B. e?mail: irene.bozzoni@uniroma1.it doi:10.1038/nrg3606Published online 3 December 2013 microRNAs (miRNAs). Small non-coding RNAs of ~22 nucleotides that are integral components of RNA-induced silencing complex (RISC) and that recognize partially complementary target mRNAs to induce translational repression, which is often linked to degradation. Among the RISC proteins, AGO binds to miRNA and mediates the repressing activity. Long non-coding RNAs: new players in cell differentiation and development Alessandro Fatica 1 and Irene Bozzoni 1–4 Abstract | Genomes of multicellular organisms are characterized by the pervasive expression of different types of non-coding RNAs (ncRNAs). Long ncRNAs (lncRNAs) belong to a novel heterogeneous class of ncRNAs that includes thousands of different species. lncRNAs have crucial roles in gene expression control during both developmental and differentiation processes, and the number of lncRNA species increases in genomes of developmentally complex organisms, which highlights the importance of RNA-based levels of control in the evolution of multicellular organisms. In this Review, we describe the function of lncRNAs in developmental processes, such as in dosage compensation, genomic imprinting, cell differentiation and organogenesis, with a particular emphasis on mammalian development. O N -C O D I N G R N A Nature Reviews Genetics | AOP , published online 3 December 2013; doi:10.1038/nrg3606 REVIEWS

长链非编码RNA与病毒和微小非编码RNA关系的研究进展

长链非编码RNA与病毒和微小非编码RNA关系的研究进展 作者：何金花， 黎毓光， HE Jin-hua， LI Yu-guang 作者单位：广东省广州市番禺区中心医院检验科,广东广州,511400 刊名： 基础医学与临床 英文刊名：Basic & Clinical Medicine 年，卷(期)：2013,33(7) 参考文献(20条) 1.Caley DP;Pink RC;Trujillano D Long noncoding RNAs,chromatin,and development 2010 2.Tim RM;Marcel ED;John SM Long non-coding RNAs:insights into functions 2009 3.Jiang Q;Wang Y;Hao Y miR2Disease:a manually curated database for microRNA deregulation in human disease 2009 4.陈勇;刘巍microRNA影响抗肿瘤药物敏感性相关研究进展[期刊论文]-临床肿瘤学杂志 2009(9) 5.Iwakiri D;Zhou L;Samanta M Epstein-Barr virus (EBV)-encoded small RNA is released from EBV-infected cells and activates signaling from Toll-like receptor 2009 6.Eilebrecht S;Pellay FX;Odenwalder P EBER2 RNA-induced transcriptome changes identify cellular processes likely targeted during Epstein Barr Virus infection 2008 7.Zhao J;Sinclair J;Houghton J Cytomegalovirus beta2.7 RNA transcript protects endothelial cells against apoptosis during ischemia/reperfusion injury 2010 8.Buck AH;Perot J;Chisholm MA Post-transcriptional regulation of miR-27 in murine cytomegalovirus infection 2010 9.Sonkoly E;Bata-Csorgo Z;Pivarcsi AI dentification and characterization of a novel,psoriasis susceptibility-related noncoding RNA gene,PRINS 2005 10.Mao YS;Sunwoo H;Zhang B Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs 2011 11.Vigneau S;Rohrlich PS;Brahic M Tmevpg1,a candidate gene for the control of Theiler's virus persistence,could be implicated in the regulation of gamma interferon[外文期刊] 2003 12.Andersson MG;Haasnoot PC;Xu N Suppression of RNA interference by adenovirus virus-associated RNA 2005 13.Xu N;Segerman B;Zhou X Adenovirus virus-associated RNAII-derived small RNAs are efficiently incorporated into the rna-induced silencing complex and associate with polyribosomes 2007 14.C Braconi;T Kogure;N Valeri microRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in hepatocellular cancer 2011 15.Katarzyna Augoff;Brian McCue;Edward F miR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triple-negative breast cancer 2012 16.Wang JY;Liu XF;Wu HC First published online:April 27,2010 CREB up-regulates long non-coding RNA,HULC expression through interaction with microRNA-372in liver cancer 2010 17.Calin GA;Liu C;Ferracin M Ultraconserved regions encoding ncRNAS are altered in human leukemias and carcinomas [外文期刊] 2007(3) 18.Cazalla D;Yario T;SeeitZ JA Down regulation of a host microRNA by a herpesvirus saimiri noncoding RNA 2010 19.Faghihi MA;Modarresi F;Khalil AM Expression of a noncoding RNA is elevated in Alzheimer's disease and drives rapid feed-forward regulation of B-secretase expression 2009 20.Christopher S New roles for large and small viral RNAs in evading host defences 2008 本文链接：https://www.wendangku.net/doc/c82295144.html,/Periodical_jcyxylc201307035.aspx