ChIRP for RNA-Protein Interactions Analysis

Noncoding RNAs (ncRNAs) are the important functional regulators of gene expression. Most ncRNAs and protein cofactors are assembled into a wide range of ribonucleoprotein complexes (RNPs) to play their functions. As an expert in the field of protein nucleic acid interaction, Creative Biolabs provides chromatin isolation services based on RNA purification (ChIRP) technology. By sequencing analysis (ChIRP-seq) technology or mass spectrometry analysis (ChIRP-MS) technology, we can also identify ncRNAs of interest (such as long non-coding RNAs, lncRNAs) and genome-binding regions bound by RNA-binding proteins (RBPs).

• ChIRP-seq

ChIRP-seq is one powerful in vivo high-throughput technology, which can enrich the discovery of RBPs and DNA. This method is to design a biotin probe that is reversely complementary to the target RNA sequence. After the target RNA is pulled down, the DNA chromosome fragments that interact with it will be attached to the streptavidin magnetic beads, and finally, pass through high-throughput sequencing quantitative and qualitative analysis of related DNA. ChIPR-seq provides the possibility to elucidate the complexity of the regulatory events of ncRNAs, and is essential for a better understanding of gene expression and disease pathogenesis.

Fig.1 Outline of the ChIRP-seq workflow.

• ChIRP-MS

ChIRP-MS is mainly based on the ChIRP-seq technology, which provides simple probe design, highly simplified protocol and comprehensive capture of interacting proteins. The invention of ChIRP-MS makes it possible to rationally design an affinity reagent for the RNA of interest through the method of antisense oligonucleotide capture. ChIRP-MS can replace the formaldehyde by UV crosslinking to study the relationship between direct binding protein and extended protein network. In addition, proteins that bind to specific regions of ncRNAs can be isolated by using antisense probes specific to the region of interest. Therefore, ChIRP-MS provides a potentially universal interaction group discovery strategy that can be easily applied to any RNA of interest.

Fig.2 Outline of the ChIRP-MS workflow. (Chu, 2015)

The Merits of ChIRP-Seq/MS

At present, ChIRP-based technologies have become increasingly favored by researchers.

• Simplification of detection: ChIRP does not require a priori knowledge of the ncRNA domains involved in the interaction, because they tile the oligonucleotide throughout the target RNA sequence, trying to cover it as much as possible;
• High throughput and large coverage: to avoid deviation from the target, ChIRP usually prefers to use probe sets for multiple regions of the target RNA;
• Wide sample range: applicable in any kinds of eukaryotic cells;
• Flexible experimental design: it can carry out corresponding detection and system analysis for different components (ncRNA, RBP and genomic DNA).

Based on our rich experience accumulation and comprehensive technology platform, Creative Biolabs provides personalized analysis services on protein-RNA interactions according to customer needs, thereby providing further strong support for the study of RNA and even gene-mediated complex regulatory mechanisms. Please do not hesitate to contact us for more details

Reference

1. Chu, C.; et al. Systematic Discovery of Xist RNA Binding Proteins. Cell. 2015, 161(2): 404-416.