Many classes of non-coding RNAs (ncRNAs), such as miRNAs and lncRNAs, have been identified as key regulators of gene expression through the interaction with proteins. CD Genomics provides ChIRP-MS, which combines in vivo RNA-interactome capture with mass spectrometry (MS), for the systematic and comprehensive identification of RNA-binding proteins (RBPs), offering important insights into RNA biology.
Overview
The interaction between ncRNAs and proteins is essential for RNA stability, localization and function. When ncRNAs are embedded in diverse ribonucleoprotein (RNP) complexes, they regulate the processes of gene expression. Mass spectrometry has emerged as a robust tool to study the ncRNA function by identifying the RBPs and measuring the effects of certain ncRNA on global protein levels. Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) is a novel technique for large-scale identification and characterization of endogenous ribonucleoprotein complexes. ChIRP-MS works excellently across a wide range of expression levels, from abundant RNAs to lowly expressed RNAs such as Xist (an essential lncRNA for X chromosome inactivation). This method has been successfully applied to comprehensively detecting the Xist-interactome.
We provide ChIRP-MS service for systematic identification and quantification of RBPs in vivo from various cell types and organism models. First, in vivo RNA–protein interactions are first chemically cross-linked. Next, chromatin is isolated and fragmented by sonication, and biotinylated oligonucleotides complementary to the complete RNA sequence of interest are added to hybridize with the target RNA. Subsequently, the hybrids, which consist of the target RNA, the chromatin and the cross-linked proteins, are isolated using streptavidin-coated beads. Finally, the hybrids are gently eluted, followed by de-cross-linking, protein extraction and MS analysis.
Features
Large Scale
Bioinformatics Analysis
High Performance
Seamless Integration
Large-scale identification and characterization of endogenous RBPs
Data QC; detection of RBPs; measure of the impact of ncRNAs on global protein levels
Optimized experimental procedures, advanced LC-MS system, expertise in operation.
Provide a full range of MS- and NGS-based methods for the study of RNA-protein interactions
Project Workflow
1. In Vivo Cross-Linking
Cells are cross-linked in vivo.
2. Cell Lysis & Sonication
Chromatin is extracted and sheared by sonication.
3. Hybridization with Probes
Biotinylated oligonucleotides complementary to the RNA of interest are hybridized with the target RNA.
4. Protein Purification
The hybrids are extracted using streptavidin-coated beads;
elution of hybrids from beads; de-cross-linking; protein extraction.
5. Mass Spectrometry
Comprehensively identify RBPs;
measure the effect of ncRNA on global protein levels.
Sample Requirements
We work with a wide range of sample types including protein solution, fresh tissue, cultured cells, blood, and microbial sample. Please feel free to contact us for sample size.
Sample Storage: extracted protein should be stored at -80°C. Avoid repeated freezing and thawing.
Shipping Method:When shipping the sample, it is stored in a 1.5 mL Eppendorf tube, sealed with a sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.
Deliverable: data QC report, MS results, integrated experimental report (materials, methodologies, and bioinformatics analysis).
References:
Chu, Ci, and Howard Y. Chang. ChIRP-MS: RNA-directed proteomic discovery. X-Chromosome Inactivation. Humana, New York, NY, 2018. 37-45.
Giambruno R, Mihailovich M, Bonaldi T. Mass spectrometry-based proteomics to unveil the Non-Coding RNA world. Frontiers in Molecular Biosciences, 2018, 5: 90.
Chu C, Zhang Q C, Da Rocha S T, et al. Systematic discovery of Xist RNA binding proteins. Cell, 2015, 161(2): 404-416.
* For Research Use Only. Not for use in diagnostic procedures.
