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.
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.
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.
Figure 2: The % input recoveries of the RNA-ChIP reactions illustrates the enrichment by Suz12 antibody.
The RNA-ChIP technique is distinct from RNA Immunoprecipita