Procedural Overview{{Citation|last1=Baroni|first1=Timothy E.|title=Advances in RIP-Chip Analysis: RNA-Binding Protein Immunoprecipitation-Microarray Profiling|date=2008|work=Post-Transcriptional Gene Regulation|pages=93–108|editor-last=Wilusz|editor-first=Jeffrey|series=Methods In Molecular Biology|place=Totowa, NJ|publisher=Humana Press|language=en|doi=10.1007/978-1-59745-033-1_6|isbn=978-1-59745-033-1|last2=Chittur|first2=Sridar V.|last3=George|first3=Ajish D.|last4=Tenenbaum|first4=Scott A.|volume=419|pmid=18369977}}{{Citation|last=Conrad|first=Nicholas K.|chapter=Chapter 15 - Co-Immunoprecipitation Techniques for Assessing RNA–Protein Interactions In Vivo|date=2008-01-01|url=http://www.sciencedirect.com/science/article/pii/S0076687908024154|series=Methods in Enzymology|volume=449|pages=317–342|editor-last=Maquat|editor-first=Lynne E.|title=RNA Turnover in Eukaryotes: Analysis of Specialized and Quality Control RNA Decay Pathways|publisher=Academic Press|doi=10.1016/S0076-6879(08)02415-4|pmid=19215765|isbn=978-0-12-374584-2 |language=en|access-date=2020-12-01|editor2-last=Kiledjian|editor2-first=Megerditch|url-access=subscription}}{{Cite journal|last1=Niranjanakumari|first1=Somashe|last2=Lasda|first2=Erika|last3=Brazas|first3=Robert|last4=Garcia-Blanco|first4=Mariano A.|date=2002-02-01|title=Reversible cross-linking combined with immunoprecipitation to study RNA–protein interactions in vivo|url=http://www.sciencedirect.com/science/article/pii/S104620230200021X|journal=Methods|language=en|volume=26|issue=2|pages=182–190|doi=10.1016/S1046-2023(02)00021-X|pmid=12054895|issn=1046-2023|url-access=subscription}}
•
Collect and lyse the cells of interest. •
Isolate all RNA fragments and the proteins bound to them from the solution. •
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. •
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. •
Disassociate the RNA from the protein of interest. •
Isolate the RNA fragments from the protein using a centrifuge. •
Use Reverse Transcription PCR to convert the RNA fragments into cDNA (DNA that is complementary to the RNA fragments). •
Fluorescently label these cDNA fragments. •
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. •
Allow the cDNA fragments to competitively hybridize to the DNA sequences bound to the chip. •
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 ==