RNA-binding proteins (RBPs) determine mRNA fate from synthesis to degradation. We provide a comprehensive and unbiased method, named mRNA interactome capture, to determine the repertoire of proteins that directly bind to mRNAs in vivo. We provide a full range of services in the study of RNA-protein interactions to offer important insights into RNA biology.
Overview
RBPs can bind to RNAs to form ribonucleoprotein complexes (RNPs), which are necessary to maintain the endogenous biological processes including post-transcriptional gene regulation and determination of RNA fate from RNA synthesis, processing, transportation, localization, translation and decay. Traditionally, the prediction of genome-wide RBPs relies on the computational analysis of protein sequence, structure and protein-RNA docking. However, in silico strategies are generally based on canonical RBDs, which could cause bias because RBPs containing non-canonical RBDs have been recently identified. A novel approach called "mRNA interactome capture", which combines UV crosslinking of RBPs to RNA and mass spectrometry (MS), is a systematic and unbiased tool for the determination and characterization of the mRNA interactome. We provide mRNA interactome capture service employing the most advanced LC-MS systems and computational strategies. The workflow entails in vivo UV crosslinking, pull down, purification of mRNAs and their binding proteins by oligo (dT) beads, protein extraction and the subsequent identification of RBPs by MS. We provide sample-to-data service to discover m RNA-bound proteomes in mammalians, plants and yeasts.
Features
Any Species
Transcriptome-Wide
Bioinformatics Analysis
Validated Process
This method can be applied to any species, from microorganisms to humans.
Profile transcriptome-wide mRNA interactome, either known or not.
Data QC; determination of mRNPs; quantitative analysis
Validated experimental process using LC-MS/MS with MRM
Project Workflow
1. UV Crosslinking
In vivo covalent bond between RNA and their interacting proteins
2. Oligo (dT) Capture
Removal of non-covalent binders using oligo (dT) magnetic beads
3. Protein Purification
Cell lysis; stringent washes; protein release by RNase treatment
4. Mass Spectrometry
Determination of the mRNA interactome; discovery of novel features of RBPs
Sample Requirements
We work with a wide range of sample types including protein solution, fresh tissue, cultured cells, blood, and microbial samples. 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).
Reference:
Castello A, Fischer B, Eichelbaum K, et al. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell, 2012, 149(6): 1393-1406.
* For Research Use Only. Not for use in diagnostic procedures.
