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RNA ChIP-IT®

クロマチンからのRNA-タンパク質複合体の単離・研究を最適化

ゲノム制御におけるRNAの役割の解析を促進するために,アクティブ・モティフ社は,RNA-ChIPに有益な最初のキットを開発するために,弊社の ChIP-IT® Express Kits キットを開発するのに用いた効果的な磁気ビーズ・ベースのプロトコールを適用しました。 RNA ChIP-IT® Kit は,クロマチンコンテクストでRNA-タンパク質相互作用を研究するようにデザインされたキットで,RT-PCR解析用のRNAを回収するために最適化されています。キットには,25反応行うのに十分な試薬と磁気ビーズを用いた最適化されたプロトコルが含まれます。

  • 特にクロマチン関連RNA研究に最適
  • RNAの完全性を維持しながら,DNAを除去するデザイン
  • RNA維持のために最適化された,クロマチン固定,超音波処理と免疫沈降の固定のための段階的なプロトコール
  • 正確な濃度のRNaseおよびプロテアーゼ阻害剤を同梱
  • コントロール抗体およびプライマーを含むコントロールキットを別売りで提供

また,RNA ChIP-IT Control Kit - Humanは,クロマチン試料が期待通りに調製されたことを確認するために,ヒト由来試料に特異的な試薬(ポジティブコントロール Suz12ウサギ・ポリクローナル抗体,ネガティブコントロールウサギIgGおよびlincSFPQ PCRプライマー)を提供します。またコントロールキットは,使用する抗体がRNA-ChIPで機能するかを確認することを簡単にします。

 
Name Format Cat No. 価格 (税抜)  
RNA ChIP-IT® 25 rxns 53024 ¥107,000 Buy Now
RNA ChIP-IT® Control Kit – Human 5 rxns 53025 ¥27,000 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.