The long non-coding RNAs (lncRNAs) are defined as transcripts with lengths greater than 200 bases that are not translated into proteins. RAP-MS is a robust approach for purifying the interaction complex of RNA in vivo and identifying the direct binders by quantitative mass spectrometry (MS). We are dedicated to delivering accurate, efficient and comprehensive data that provides detailed insights into the lncRNA biology, even with partially degraded RNA samples.

Overview

The interactions between non-coding RNAs (ncRNAs) and proteins are very critical for ncRNA stability, localization, and function. The lncRNA is an important class of ncRNAs that are required for the regulation of gene expression in cellular differentiation, development and human diseases, as well as the transcriptional silencing of one X chromosome, in a process known as X-chromosome inactivation (XCI) that occurs in mammalian female cells. RAP (RNA antisense purification) uses long (>60 nucleotides) biotinylated antisense probes, which form very stable RNA-DNA hybrids, thus permitting the purification of lncRNA complexes in denaturing and reducing conditions. The subsequent UV crosslinking creates covalent bonds exclusively between directly interacting RNA and proteins. The employ of stable isotope labeling by amino acids in culture (SILAC)-based MS achieves better discrimination of specific associations from background proteins. All these steps make RAP-MS the elective strategy for the combined identification of the DNA-binding sites and protein-interactors of lncRNAs such as Xist-interacting proteins that play a role in XCI. RAP-MS provides a short list of high confidence protein interactors (RBPs), which is more efficient than previous strategies such as CLIP (crosslinking and immunoprecipitation)-based methods. CLIP methods are more used for identifying novel protein interaction partners for a target ncRNA.

Project Workflow

Sample Requirements

We work with a wide range of sample types including protein solution, fresh tissue, cultured cells, blood, and microbial colonies. 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:

1. Giambruno R, Mihailovich M, Bonaldi T. Mass spectrometry-based proteomics to unveil the Non-Coding RNA world. Frontiers in Molecular Biosciences, 2018, 5: 90.
2. Zlotorynski E. Non-coding RNA: X-chromosome inactivation unravelled. Nature Reviews Molecular Cell Biology, 2015, 16(6): 325.
3. McHugh, Colleen A., and Mitchell Guttman. RAP-MS: a method to identify proteins that interact directly with a specific RNA molecule in cells. RNA Detection. Humana Press, New York, NY, 2018. 473-488.