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RNA ChIP-IT®

optimized to isolate and study RNA-protein complexes specifically from chromatin

To facilitate the characterization of the role of RNA in genome regulation, Active Motif has applied the efficient magnetic bead-based protocol used to develop our ChIP-IT® Express Kits to develop the first of its kind kit for RNA-ChIP. The RNA ChIP-IT® Kit was designed to study RNA-protein interactions in a chromatin context, and has been optimized to recover RNA for RT-PCR analysis. The kit contains sufficient material for 25 assays, with optimized protocols using magnetic beads, to minimize labor required to obtain results.

  • Specifically tailored to study chromatin-associated RNA
  • Designed to remove DNA while maintaining RNA integrity
  • Step-by-step protocols for fixation of chromatin, sonication and immunoprecipitation, all optimized for RNA preservation
  • Includes all RNase and protease inhibitors at precise concentrations
  • Separate control kit available with control antibody and primers

Also available is the RNA ChIP-IT Control Kit – Human, which provides human-specific reagents (positive control Suz12 rabbit polyclonal antibody, negative control rabbit IgG and lincSFPQ PCR primer mix) to verify that the chromatin samples prepared by the user perform as expected. The control kit also make it easier to validate if your antibodies function in RNA-ChIP.

 
Name Format Cat No. Price  
RNA ChIP-IT® 25 rxns 53024 $570 Buy Now
RNA ChIP-IT® Control Kit – Human 5 rxns 53025 $120 Buy Now

Evidence is building that RNA-directed processes play a critical role in orchestrating chromatin architecture and epigenetic memory. Nucleic acids purified from chromatin are 2-5% RNA; these RNAs are non-coding sequences that play important roles in chromatin structure and transcriptional silencing. But, characterizing these RNAs by ChIP techniques is complicated by the complexity of chromatin structure, the difficulty of working with RNA, and the high amounts of DNA in chromatin. RNA ChIP-IT uses a modified ChIP protocol that has been optimized for RNA preservation and recovery. RNA-protein interactions are fixed with formaldehyde, and chromatin shearing is combined with DNase treatment to yield RNA/protein complexes that can be immunoprecipitated with antibodies to specific proteins. Cross-links are subsequently reversed; RNA is recovered and again treated with DNase to ensure the absence of DNA. The optimized method is quick and has been successfully used to study several non-coding RNAs in the chromatin context.

Real-time RT-PCR analysis of Suz12/normal rabbit IgG RNA-ChIP samples
Figure 1: Real-time RT-PCR analysis of Suz12/normal rabbit IgG RNA-ChIP samples.

The RNA ChIP-IT Kit was used on 10 µg samples of DNase I-treated HeLa chromatin with 10 µl of Suz12 antibody (Cat. No. 39357) and 2 µg of Normal rabbit IgG. The RNA-IP was performed overnight at 4°C. Real-time RT-PCR was performed using primers for the lincRNA SFPQ locus. The amplification plot is shown.

 
Real-time RT-PCR analysis of Suz12/normal rabbit IgG RNA-ChIP samples.
Figure 2: The % input recoveries of the RNA-ChIP reactions illustrates the enrichment by Suz12 antibody.

The RNA-ChIP technique is distinct from RNA Immunoprecipitation, or RIP, which is sometimes used to study RNA-protein interactions outside of the context of chromatin. Chromatin-associated RNAs must be separated from formaldehyde-fixed chromatin before immunoprecipitation. The Active Motif RNA ChIP-IT kit has optimized all the procedures required, so you can proceed with your experiments.

 
Flow chart of the RNA ChIP-IT method to perform chromatin IP of RNA.
 
Figure 1: Flow chart of the RNA ChIP-IT method.
 

RNA ChIP-IT is a comprehensive kit for performing RNA-ChIP

RNA ChIP-IT Kits provide sufficient components to perform 25 ChIP reactions. Sufficient shearing components are included to optimize shearing conditions and then make 5 preparations of RNA-bearing sheared chromatin from three 15 cm plates (4.5 x 107cells); each of these chromatin preparations yield enough material to perform approximately 16 RNA ChIP assays. We recommend that you also purchase an RNA ChIP-IT Control Kit – Human, as the use of validated controls helps in troubleshooting, interpreting your results and validating antibodies for use in ChIP.

Contents & Storage

Protein G Magnetic Beads, Bar Magnet, Sticky Dots, Shearing Buffer (or Digestion Buffer and Enzymatic Shearing Cocktail),  DEPC Water, RNase Inhibitors, DNase I, DNA Digestion Buffer, 5M NaCl, RNA-IP Buffer, RNA-ChIP Wash buffers, RNA-ChIP Elution Buffer, 10X DNase I Reaction Buffer, DNase I Stop Solution, 10X PBS, 10X Glycine, 1X Lysis Buffer, Protease Inhibitor Cocktail, PMSF and EDTA. Reagent storage conditions vary from room temperature to -20°C. Please download a manual for complete kit specifications and storage conditions. All reagents are guaranteed stable for 6 months when stored properly.