EpiNext CUT&LUNCH RNA Immunoprecipitation (RIP) Kit

The EpiNext™ CUT&LUNCH (Cleavage Under Target and Liberate Unique Nucleic Complex Homogeneously) RNA Immunoprecipitation (RIP) Kit provides a rapid and efficient method for studying in vivo protein-RNA interactions. It integrates all the advantages of the currently used RIP and modified CUT&RUN, a new method for protein-DNA interaction profiling. The CUT&LUNCH RIP Kit has the following advantages and features:

• Extremely Fast 3-Step Protocol: Start directly from intact cells without needing lysis and crosslinking. The entire procedure of the assay can be finished within 1 h and 50 min, minimizing RNA damage and loss of disassociated RNA binding components and preserving native protein-RNA structure.
• Enhanced Specificity and Minimized Background: Unique nucleic acid cleavage enzymes ensure low sequence bias by cleaving or removing RNA sequences at both ends of the target protein/RNA complex without affecting RNA occupied by the target protein. The liberated non-specific protein-RNA complexes are selectively eliminated, allowing for the recovery of only the antibody-bound complexes. As a result, target protein-enriched regions can be reliably achieved and identified through high-resolution mapping.
• Low Input Materials: Robust unbound RNA cleavage and immunocapture are all processed in the same single tube. This method uses both cells and tissues and allows for maximal degradation protection of the target protein with minimal sample loss. As a result, the input cell amount can be as few as 20,000 cells, <5% of the minimal amount required by traditional RIP.
• Wide Suitability: Ideal for cultured cells as well as fresh and frozen tissues from various species for different RNA binding proteins while maintaining both assay specificity and sensitivity.
• Highly Convenient The kit contains all required components, making the EpiNext™ CUT&LUNCH RNA Immunoprecipitation (RIP) Kit both convenient and cost-effective.
  

Background Information

Enrichment of protein-complexed RNA in vivo followed by qPCR and/or next-generation sequencing offers an advantageous tool for studying epitranscriptome-wide protein-RNA interactions. The major methods used for a long time to achieve this goal are traditional RIP (RNA immunoprecipitation) followed by PCR (RIP-PCR) or sequencing (RIP-Seq). These methods have been widely used but are unable to achieve high resolution, need crosslinking, and suffer from poor reproducibility and a complicated process. In particular, these methods are time-consuming (from 8 hours to 2 days) and costly.

Principle & Procedure

This kit provides all necessary reagents to enrich RNA binding protein-specific RNA complexes, enabling profiling of protein-RNA interactions from various cell samples via qRT-PCR or NGS. In this assay, cells are permeabilized and treated with a RIP-grade antibody specific to the protein of interest. A unique nucleic acid cleavage enzyme mix then selectively cleaves RNA sequences flanking the target protein regions, removing non-specific RNA-protein complexes. Only antibody-bound complexes are selectively retained. The RNA fragments in the captured protein/RNA complexes are purified and can be directly used for qRT-PCR or cDNA library construction to profile interactions between proteins and RNA.

Starting Materials

Starting materials can include various mammalian cell samples such as culture cells from a flask or plate, primary cells, or rare cell populations isolated from blood, body fluid, fresh/frozen tissues, and specific cells sorted from entire cell populations and embryonic cells, etc.