RIP-chip (RNA immunoprecipitation chip) is a

molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...

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 sam ...

with a

microarray A microarray is a multiplex (assay), multiplex lab-on-a-chip. Its purpose is to simultaneously detect the expression of thousands of biological interactions. It is a two-dimensional array on a Substrate (materials science), solid substrate—usu ...

. The purpose of this technique is to identify which

RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...

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, an ...

. It can also be used to determine relative levels of

gene expression Gene expression is the process (including its Regulation of gene expression, regulation) by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, ...

, 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) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as bacteria and viruses, including those that caus ...

. 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 subject a specimen to a specified constant force - for example, to separate various components of a fluid. This is achieved by spinning the fluid at high speed within a container, thereby ...

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 that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...

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

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

. 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 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 genes, other functional regions of the genome such as ...

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 followed by RT-PCR, however such selective analysis cannot be used when the bound RNAs are not yet known. 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, thymine, cytosine, and guanine. 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 technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also kn ...

. 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, thymine, cytosine, and guanine. The ...

platform, Illumina. * CLIP: The RNA binding protein is cross-linked to the RNA via the use of UV light prior to lysis, which is followed by RNA fragmentation, immunoprecipitation, high-salt wash, SDS-PAGE, membrane transfer, proteinase digestion, cDNA library preparation and sequencing in order to identify the direct RNA binding sites. CLIP has first been combined with high throughput sequencing in

HITS-CLIP High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) is a variant of CLIP for genome-wide mapping protein–RNA binding sites or RNA modification sites in vivo. HITS-CLIP was originally used to generate ge ...

to determine Nova-RNA binding sites in the mouse brain, and in

iCLIP iCLIP (individual-nucleotide resolution crossLinking and immunoprecipitation) 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 RN ...

that enabled amplification of truncated cDNAs and introduced the use of UMIs. *

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