RIP-chip (RNA immunoprecipitation chip) is a

molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...

technique which combines RNA

immunoprecipitation 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 samp ...

with a microarray. The purpose of this technique is to identify which

RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

sequences interact with a particular

RNA binding protein RNA-binding proteins (often abbreviated as RBPs) are proteins that bind to the double or single stranded RNA in cells and participate in forming ribonucleoprotein complexes. RBPs contain various structural motifs, such as RNA recognition motif ...

of interest

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...

. It can also be used to determine relative levels of

gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...

, to identify subsets of RNAs which may be co-regulated, or to identify RNAs that may have related functions. This technique provides insight into the post-transcriptional gene regulation which occurs between RNA and RNA binding proteins.

Procedural Overview

# 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 An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

. 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 A centrifuge is a device that uses centrifugal force to separate various components of a fluid. This is achieved by spinning the fluid at high speed within a container, thereby separating fluids of different densities (e.g. cream from milk) or ...

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 In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...

(DNA that is complementary to the RNA fragments). # Fluorescently label these cDNA fragments. # Prepare the

gene chip A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to ...

. 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 In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...

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

Previous techniques aiming to understand protein-RNA interactions included RNA Electrophoretic Mobility Shift Assays and UV-crosslinking, however these techniques cannot be used when the RNA sequence is unknown. 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. Alternatives to RIP-chip include: * RIP-seq: Involves sequencing the RNAs that were pulled down using

high-throughput sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The ...

rather than analyzing them with a microarray. Authors Zhao et al., 2010. combined the RNA immunoprecipitation procedure with

RNA sequencing RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing ...

. Using specific antibodies (α-Ezh2) they immunoprecipitated nuclear RNA isolated from mouse ES cells, and subsequently sequenced the pulled-down RNA using the

next generation sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The ...

platform, Illumina. * CLIP-seq: The RNA binding protein is cross-linked to the RNA via the use of UV light prior to immunoprecipitation. Authors Licatalosi et al., 2008 first combined the UV crosslinking coupled immunoprecipitation procedure ( CLIP) with high throughput sequencing methods to determine Nova-RNA binding sites in the mouse brain. In addition, they found that this protocol could determine ''de novo'' protein interactions. *

ChIP-on-chip ChIP-on-chip (also known as ChIP-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' ...

: A similar technique which detects the binding of proteins to genomic DNA rather than RNA.

References

{{Reflist Genetics techniques Microarrays RNA Protein methods