RNA immunoprecipitation chip

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RIP-chip (RNA immunoprecipitation chip) is a molecular biology technique which combines RNA immunoprecipitation with a microarray. The purpose of this technique is to identify which RNA sequences interact with a particular RNA binding protein of interest in vivo. [1] [2] [3] [4] It can also be used to determine relative levels of gene expression, to identify subsets of RNAs which may be co-regulated, or to identify RNAs that may have related functions. [4] [5] This technique provides insight into the post-transcriptional gene regulation which occurs between RNA and RNA binding proteins. [5]

Contents

An overview of the major stages in the RNA Immunoprecipitation chip procedure. RIP-chip Overview.pdf
An overview of the major stages in the RNA Immunoprecipitation chip procedure.

Procedural Overview [6] [7] [8]

  1. Collect and lyse the cells of interest.
  2. Isolate all RNA fragments and the proteins bound to them from the solution.
  3. Immunoprecipitate the protein of interest. The solution containing the protein-bound RNAs is washed over beads which have been conjugated to antibodies. These antibodies are designed to bind to the protein of interest. They pull the protein (and any RNA fragments that are specifically bound to it) out of the solution which contains the rest of the cell contents.
  4. Dissociate the protein-bound RNA from the antibody-bead complex. Then, use a centrifuge to separate the protein-bound RNA from the heavier antibody-bead complexes, keeping the protein-bound RNA and discarding the beads.
  5. Disassociate the RNA from the protein of interest.
  6. Isolate the RNA fragments from the protein using a centrifuge.
  7. Use Reverse Transcription PCR to convert the RNA fragments into cDNA (DNA that is complementary to the RNA fragments).
  8. Fluorescently label these cDNA fragments.
  9. Prepare the gene chip . This is a small chip that has DNA sequences bound to it in known locations. These DNA sequences correspond to all of the known genes in the genome of the organism that the researcher is working with (or a subset of genes that the researcher is interested in). The cDNA sequences that have been collected will be complementary to some of these DNA sequences, as the cDNAs represent a subset of the RNAs transcribed from the genome.
  10. Allow the cDNA fragments to competitively hybridize to the DNA sequences bound to the chip.
  11. Detection of the fluorescent signal from the cDNA bound to the chip tells researchers which gene(s) on the chip were hybridized to the cDNA.

The genes fluorescently identified by the chip analysis are the genes whose RNA interacts with the original protein of interest. The strength of the fluorescent signal for a particular gene can indicate how much of that particular RNA was present in the original sample, which indicates the expression level of that gene.

Development and Similar Techniques

Previous techniques aiming to understand protein-RNA interactions included RNA Electrophoretic Mobility Shift Assays and UV-crosslinking followed by RT-PCR, [9] however such selective analysis cannot be used when the bound RNAs are not yet known. [1] To resolve this, RIP-chip combines RNA immunoprecipitation to isolate RNA molecules interacting with specific proteins with a microarray which can elucidate the identity of the RNAs participating in this interaction. [5] [1] Alternatives to RIP-chip include:

Related Research Articles

Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure.

<span class="mw-page-title-main">Regulator gene</span>

A regulator gene, regulator, or regulatory gene is a gene involved in controlling the expression of one or more other genes. Regulatory sequences, which encode regulatory genes, are often at the five prime end (5') to the start site of transcription of the gene they regulate. In addition, these sequences can also be found at the three prime end (3') to the transcription start site. In both cases, whether the regulatory sequence occurs before (5') or after (3') the gene it regulates, the sequence is often many kilobases away from the transcription start site. A regulator gene may encode a protein, or it may work at the level of RNA, as in the case of genes encoding microRNAs. An example of a regulator gene is a gene that codes for a repressor protein that inhibits the activity of an operator.

Cross-linking and immunoprecipitation is a method used in molecular biology that combines UV crosslinking with immunoprecipitation in order to identify RNA binding sites of proteins on a transcriptome-wide scale, thereby increasing our understanding of post-transcriptional regulatory networks. CLIP can be used either with antibodies against endogenous proteins, or with common peptide tags or affinity purification, which enables the possibility of profiling model organisms or RBPs otherwise lacking suitable antibodies.

<span class="mw-page-title-main">Nuclear run-on</span>

A nuclear run-on assay is conducted to identify the genes that are being transcribed at a certain time point. Approximately one million cell nuclei are isolated and incubated with labeled nucleotides, and genes in the process of being transcribed are detected by hybridization of extracted RNA to gene specific probes on a blot. Garcia-Martinez et al. (2004) developed a protocol for the yeast S. cerevisiae that allows for the calculation of transcription rates (TRs) for all yeast genes to estimate mRNA stabilities for all yeast mRNAs.

<span class="mw-page-title-main">RNA spike-in</span>

An RNA spike-in is an RNA transcript of known sequence and quantity used to calibrate measurements in RNA hybridization assays, such as DNA microarray experiments, RT-qPCR, and RNA-Seq.

<span class="mw-page-title-main">ChIP-on-chip</span> Molecular biology method

ChIP-on-chip is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray ("chip"). Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo. Specifically, it allows the identification of the cistrome, the sum of binding sites, for DNA-binding proteins on a genome-wide basis. Whole-genome analysis can be performed to determine the locations of binding sites for almost any protein of interest. As the name of the technique suggests, such proteins are generally those operating in the context of chromatin. The most prominent representatives of this class are transcription factors, replication-related proteins, like origin recognition complex protein (ORC), histones, their variants, and histone modifications.

ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest. Previously, ChIP-on-chip was the most common technique utilized to study these protein–DNA relations.

<span class="mw-page-title-main">Tiling array</span>

Tiling arrays are a subtype of microarray chips. Like traditional microarrays, they function by hybridizing labeled DNA or RNA target molecules to probes fixed onto a solid surface.

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.

Methylated DNA immunoprecipitation is a large-scale purification technique in molecular biology that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine (5mC). This technique was first described by Weber M. et al. in 2005 and has helped pave the way for viable methylome-level assessment efforts, as the purified fraction of methylated DNA can be input to high-throughput DNA detection methods such as high-resolution DNA microarrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). Nonetheless, understanding of the methylome remains rudimentary; its study is complicated by the fact that, like other epigenetic properties, patterns vary from cell-type to cell-type.

<span class="mw-page-title-main">Chromatin immunoprecipitation</span> Genomic technique

Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly define cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. ChIP is crucial for the advancements in the field of epigenomics and learning more about epigenetic phenomena.

<span class="mw-page-title-main">ChIP-exo</span>

ChIP-exo is a chromatin immunoprecipitation based method for mapping the locations at which a protein of interest binds to the genome. It is a modification of the ChIP-seq protocol, improving the resolution of binding sites from hundreds of base pairs to almost one base pair. It employs the use of exonucleases to degrade strands of the protein-bound DNA in the 5'-3' direction to within a small number of nucleotides of the protein binding site. The nucleotides of the exonuclease-treated ends are determined using some combination of DNA sequencing, microarrays, and PCR. These sequences are then mapped to the genome to identify the locations on the genome at which the protein binds.

iCLIP is a variant of the original CLIP method used for identifying protein-RNA interactions, which uses UV light to covalently bind proteins and RNA molecules to identify RNA binding sites of proteins. This crosslinking step has generally less background than standard RNA immunoprecipitation (RIP) protocols, because the covalent bond formed by UV light allows RNA to be fragmented, followed by stringent purification, and this also enables CLIP to identify the positions of protein-RNA interactions. As with all CLIP methods, iCLIP allows for a very stringent purification of the linked protein-RNA complexes by stringent washing during immunoprecipitation followed by SDS-PAGE and transfer to nitrocellulose. The labelled protein-RNA complexes are then visualised for quality control, excised from nitrocellulose, and treated with proteinase to release the RNA, leaving only a few amino acids at the crosslink site of the RNA.

DRIP-seq (DRIP-sequencing) is a technology for genome-wide profiling of a type of DNA-RNA hybrid called an "R-loop". DRIP-seq utilizes a sequence-independent but structure-specific antibody for DNA-RNA immunoprecipitation (DRIP) to capture R-loops for massively parallel DNA sequencing.

<span class="mw-page-title-main">Epitranscriptomic sequencing</span>

In epitranscriptomic sequencing, most methods focus on either (1) enrichment and purification of the modified RNA molecules before running on the RNA sequencer, or (2) improving or modifying bioinformatics analysis pipelines to call the modification peaks. Most methods have been adapted and optimized for mRNA molecules, except for modified bisulfite sequencing for profiling 5-methylcytidine which was optimized for tRNAs and rRNAs.

Selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq) is a technique developed for the rapid identification of DNA binding specificities and affinities of full length monomeric and dimeric transcription factors in a fast and semi-high-throughput fashion.

CUT&RUN sequencing, also known as cleavage under targets and release using nuclease, is a method used to analyze protein interactions with DNA. CUT&RUN sequencing combines antibody-targeted controlled cleavage by micrococcal nuclease with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN sequencing does not.

CUT&Tag-sequencing, also known as cleavage under targets and tagmentation, is a method used to analyze protein interactions with DNA. CUT&Tag-sequencing combines antibody-targeted controlled cleavage by a protein A-Tn5 fusion with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN and CUT&Tag sequencing do not. CUT&Tag sequencing is an improvement over CUT&RUN because it does not require cells to be lysed or chromatin to be fractionated. CUT&RUN is not suitable for single-cell platforms so CUT&Tag is advantageous for these.

ChIL sequencing (ChIL-seq), also known as Chromatin Integration Labeling sequencing, is a method used to analyze protein interactions with DNA. ChIL-sequencing combines antibody-targeted controlled cleavage by Tn5 transposase with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that ChIL-Sequencing does not. ChIL-Seq is a precise technique that reduces sample loss could be applied to single-cells.

<span class="mw-page-title-main">Ribonucleoprotein Networks Analyzed by Mutational Profiling</span> Protein-RNA binding probing method

Ribonucleoprotein Networks Analyzed by Mutational Profiling (RNP-MaP) is a strategy for probing RNA-protein networks and protein binding sites at a nucleotide resolution. Information about RNP assembly and function can facilitate a better understanding of biological mechanisms. RNP-MaP uses NHS-diazirine (SDA), a hetero-bifunctional crosslinker, to freeze RNA-bound proteins in place. Once the RNA-protein crosslinks are formed, MaP reverse transcription is then conducted to reversely transcribe the protein-bound RNAs as well as introduce mutations at the site of RNA-protein crosslinks. Sequencing results of the cDNAs reveal information about both protein-RNA interaction networks and protein binding sites.

References

  1. 1 2 3 Gagliardi, Miriam; Matarazzo, Maria R. (2016), Lanzuolo, Chiara; Bodega, Beatrice (eds.), "RIP: RNA Immunoprecipitation", Polycomb Group Proteins: Methods and Protocols, Methods in Molecular Biology, New York, NY: Springer, vol. 1480, pp. 73–86, doi:10.1007/978-1-4939-6380-5_7, ISBN   978-1-4939-6380-5, PMID   27659976 , retrieved 2020-12-01
  2. ↑ Townley-Tilson, W. H. Davin; Pendergrass, Sarah A.; Marzluff, William F.; Whitfield, Michael L. (2006-10-01). "Genome-wide analysis of mRNAs bound to the histone stem–loop binding protein". RNA. 12 (10): 1853–1867. doi:10.1261/rna.76006. ISSN   1355-8382. PMC   1581977 . PMID   16931877.
  3. ↑ Khalil, Ahmad M.; Guttman, Mitchell; Huarte, Maite; Garber, Manuel; Raj, Arjun; Morales, Dianali Rivea; Thomas, Kelly; Presser, Aviva; Bernstein, Bradley E.; Oudenaarden, Alexander van; Regev, Aviv (2009-07-14). "Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression". Proceedings of the National Academy of Sciences. 106 (28): 11667–11672. Bibcode:2009PNAS..10611667K. doi: 10.1073/pnas.0904715106 . ISSN   0027-8424. PMC   2704857 . PMID   19571010.
  4. 1 2 Hendrickson, David G.; Hogan, Daniel J.; Herschlag, Daniel; Ferrell, James E.; Brown, Patrick O. (2008-05-07). "Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance". PLOS ONE. 3 (5): e2126. Bibcode:2008PLoSO...3.2126H. doi: 10.1371/journal.pone.0002126 . ISSN   1932-6203. PMC   2330160 . PMID   18461144.
  5. 1 2 3 4 Jayaseelan, Sabarinath; Doyle, Francis; Tenenbaum, Scott A. (2014-05-01). "Profiling post-transcriptionally networked mRNA subsets using RIP-Chip and RIP-Seq". Methods. Genomic approaches for studying transcriptional and post-transcriptional processes. 67 (1): 13–19. doi:10.1016/j.ymeth.2013.11.001. ISSN   1046-2023. PMC   4004666 . PMID   24257445.
  6. ↑ Baroni, Timothy E.; Chittur, Sridar V.; George, Ajish D.; Tenenbaum, Scott A. (2008), Wilusz, Jeffrey (ed.), "Advances in RIP-Chip Analysis: RNA-Binding Protein Immunoprecipitation-Microarray Profiling", Post-Transcriptional Gene Regulation, Methods In Molecular Biology, Totowa, NJ: Humana Press, vol. 419, pp. 93–108, doi:10.1007/978-1-59745-033-1_6, ISBN   978-1-59745-033-1, PMID   18369977 , retrieved 2020-12-01
  7. ↑ Conrad, Nicholas K. (2008-01-01), "Chapter 15 - Co-Immunoprecipitation Techniques for Assessing RNA–Protein Interactions In Vivo", in Maquat, Lynne E.; Kiledjian, Megerditch (eds.), RNA Turnover in Eukaryotes: Analysis of Specialized and Quality Control RNA Decay Pathways, Methods in Enzymology, vol. 449, Academic Press, pp. 317–342, doi:10.1016/S0076-6879(08)02415-4, ISBN   9780123745842, PMID   19215765 , retrieved 2020-12-01
  8. ↑ Niranjanakumari, Somashe; Lasda, Erika; Brazas, Robert; Garcia-Blanco, Mariano A. (2002-02-01). "Reversible cross-linking combined with immunoprecipitation to study RNA–protein interactions in vivo". Methods. 26 (2): 182–190. doi:10.1016/S1046-2023(02)00021-X. ISSN   1046-2023. PMID   12054895.
  9. ↑ Thomson, A.m.; Rogers, J.t.; Walker, C.e.; Staton, J.m.; Leedman, P.j. (1999-11-01). "Optimized RNA Gel-Shift and UV Cross-Linking Assays for Characterization of Cytoplasmic RNA-Protein Interactions". BioTechniques. 27 (5): 1032–1042. doi: 10.2144/99275rr03 . ISSN   0736-6205. PMID   10572651.
  10. 1 2 Zhao, Jing; Ohsumi, Toshiro K.; Kung, Johnny T.; Ogawa, Yuya; Grau, Daniel J.; Sarma, Kavitha; Song, Ji Joon; Kingston, Robert E.; Borowsky, Mark; Lee, Jeannie T. (December 2010). "Genome-wide Identification of Polycomb-Associated RNAs by RIP-seq". Molecular Cell. 40 (6): 939–953. doi:10.1016/j.molcel.2010.12.011. ISSN   1097-2765. PMC   3021903 . PMID   21172659.
  11. ↑ Ule, Jernej; Jensen, Kirk B.; Ruggiu, Matteo; Mele, Aldo; Ule, Aljaz; Darnell, Robert B. (2003-11-14). "CLIP identifies Nova-regulated RNA networks in the brain". Science. 302 (5648): 1212–1215. Bibcode:2003Sci...302.1212U. doi:10.1126/science.1090095. ISSN   1095-9203. PMID   14615540. S2CID   23420615.
  12. ↑ Licatalosi, Donny D.; Mele, Aldo; Fak, John J.; Ule, Jernej; Kayikci, Melis; Chi, Sung Wook; Clark, Tyson A.; Schweitzer, Anthony C.; Blume, John E.; Wang, Xuning; Darnell, Jennifer C. (November 2008). "HITS-CLIP yields genome-wide insights into brain alternative RNA processing". Nature. 456 (7221): 464–469. Bibcode:2008Natur.456..464L. doi:10.1038/nature07488. ISSN   1476-4687. PMC   2597294 . PMID   18978773.
  13. ↑ König, Julian; Zarnack, Kathi; Rot, Gregor; Curk, Tomaz; Kayikci, Melis; Zupan, Blaz; Turner, Daniel J.; Luscombe, Nicholas M.; Ule, Jernej (July 2010). "iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution". Nature Structural & Molecular Biology. 17 (7): 909–915. doi:10.1038/nsmb.1838. ISSN   1545-9985. PMC   3000544 . PMID   20601959.
  14. ↑ Ansorge, Wilhelm J. (2010-01-01), Patrinos, George P.; Ansorge, Wilhelm J. (eds.), "Chapter 24 - Novel Next-Generation DNA Sequencing Techniques for Ultra High-Throughput Applications in Bio-Medicine", Molecular Diagnostics (Second Edition), San Diego: Academic Press, pp. 365–378, doi:10.1016/b978-0-12-374537-8.00024-9, ISBN   978-0-12-374537-8 , retrieved 2020-12-01
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