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ChIPデータの標準化

本当の生物学的変化を明らかにするには不要なバリエーションを修正する必要があります


ChIP Normalization reveals changes in H3K27me3 levels following treatment with EZH2 inhibitor.
ChIP データ標準は,EZH2阻害剤によって生じるヒストンH3K27me3の減少を明らかにする。

アクティブ・モティフ社の ChIP スパイク・イン標準化手法 は、技術的な変化と試料処理時のバイアスの影響を低減するためのChIP qPCRChIP-Seqデータを標準化するために使用することができます。ChIPは,試料の損失,シーケンシングリードの深度の違いやユーザーの違いによるばらつきのため,結果の解釈が困難です。アクティブ・モティフ社の標準化手法は,阻害化合物,突然変異または疾患といった病因によって誘導される,真の生物学的差異を明らかにするために,バイアスを除去した上で,試料と抗体の反応に適用されます。

ChIPの標準化は,弊社のSpike-in試薬を研究者の標準的なChIPプロトコールに統合することによって,簡単に実現することができます。標準的なChIP反応は,クロマチンと目的の抗体を使用して行われます。そこに Spike-in クロマチンSpike-in 抗体 が免疫沈降反応の微量画分として加えられます。ChIPの反応中に導入された変化は,Spike-inクロマチンでも発生します。Spike-inクロマチンはすべての試料で一貫しているため,標準化係数は、Spike-inシグナルに基づいて作成され、試料のゲノムに適用されます。

ChIP スパイク・イン 標準化手法の利点

  • 技術的差異による影響の削減
  • 標準的なChIP解析で観察されていない,潜在的または微妙な生物学的な差異を検出
  • バイアスなしに異なる試料‐抗体反応に適用可能
  • Spike-inクロマチンとSpike-in 抗体は,各研究者のChIPプロトコールで使用可能
  • ChIP qPCR ChIP-Seqのいずれでも利用可能

ChIP標準化手法の詳細については,以下の Method, Data または Contents タブをクリックしてください。 マニュアルまたは関連書類については,Documents タブをクリックしてください。

 
Name Format Cat No. 価格 (税抜)  
Spike-in Antibody 50 µg 61686 ¥40,500 Buy Now
Spike-in Chromatin 15 rxns 53083 ¥35,000 Buy Now
Drosophila Positive Control Primer Set Pbgs 96 rxns 71037 ¥20,000 Buy Now
Drosophila Negative Control Primer Set 1 96 rxns 71028 ¥20,000 Buy Now
Drosophila Negative Control Primer Set 3 96 rxns 71038 ¥20,000 Buy Now

ChIP Spike-in Normalization Advantages

  • Enables normalization of ChIP results to reduce the effects of technical variation
  • Detect subtle biological differences that are not observed with standard ChIP analysis
  • Apply normalization strategy across different antibodies and samples without bias
  • Spike-in Chromatin and Spike-in Antibody can be used with any ChIP protocol
  • Normalization strategy works with both qPCR and ChIP-Seq analysis

How does the ChIP Spike-in Normalization Strategy work?

A standard ChIP reaction is set up using experimental chromatin and an antibody of interest. Spike-in chromatin and a Spike-in Antibody are also added to the reaction. The Spike-in Antibody recognizes a histone variant that is specific to the species of the Spike-in chromatin (Drosophila). This enables specific detection of the spike-in chromatin without any significant increase in background signal. Since variation introduced during the ChIP procedure will also occur with the spike-in chromatin, a normalization factor can be created from the spike-in chromatin and applied to the experimental chromatin to normalize out technical variation and sample bias, or to monitor biological effects.

  

  Sample Chromatin Spike-in Chromatin Antibody of Interest Spike-in Antibody
ChIP Spike-in Reaction Guidelines
Robust antibodies against abundant histone modifications  25 µg  Refer to lot-specific data sheet  4 µg  2 µg
Antibodies against transcription factors, histone modifiers or low abundance histone modifications  25 µg  Refer to lot-specific data sheet  4 µg  2 µg

 

Flow Chart of the ChIP Normalization Strategy for ChIP-Seq from Active Motif
Figure 1: ChIP-Seq Normalization Workflow.

A standard ChIP reaction is set up using experimental chromatin (e.g. human) and an antibody of interest. In addition, Drosophila melanogaster chromatin is added, or "spiked-in" to each reaction as a minor fraction of the total chromatin. An antibody that recognizes the Drosophila-specific histone variant, H2Av, is added to the reaction. The Spike-in antibody provides a mechnaism to reliable pull down a small fraction of the Drosophila chromatin that is consistent across all samples. Following ChIP sequencing, the the data is mapped to both the Drosophila genome and the experimental genome. A normalization factor is created for each sample based on the Drosophila tag counts. The experimental tag counts are normalized by the same factor.

Reduce the Effects of Technical Variation

The ChIP Normalization Strategy is ideal to correct for differences that results from sample loss, amplification bias, uneven sequencing read depth or hand-to-hand differences between users. By utilizing the differences observed between samples with the Spike-in chromatin, a normalization factor is created and applied to the experimental samples to normalize out the effects of technical variation.

ChIP qPCR analysis using the Spike-in chromatin
ChIP qPCR analysis using human chromatin
Normalized ChIP qPCR data using the Spike-in Normalization Strategy
Figure 1: ChIP Normalization of technical variation by ChIP qPCR.

A ChIP qPCR reaction was set up with 750 ng of Spike-in chromatin added to 30 µg of human chromatin. Both Spike-in Antibody (2 µg) and a Histone H3K27me3 antibody (4 µg) were also added to the ChIP reaction. ChIP was performed according to Active Motif's ChIP-IT High Sensitivity protocol (Catalog No. 53040), however, one sample was left as normal, one sample was given only 50% of the Protein G beads and the third sample had 50% of the IP volume removed. Enriched DNA was analyzed by qPCR using both Drosophila-specific primers and Human-specific primers for each sample. The results show the effects of these sample differences on the enrichment. The Drosophila data was used to create a normalization factor for each sample. This normalization factor was then applied to each Human data point. The normalized data reveals comparable ChIP qPCR results following the removal of technical variation effects, validating the Spike-in strategy for normalization.


Identify Biological Differences Not Observed by Standard ChIP Analysis

By adding Spike-in Chromatin and Spike-in Antibody to standard ChIP reactions, experimental data can be normalized for sample variation. This normalization makes it easier to monitor the effects of experimental conditions, such as inhibitory compounds or mutants to reveal biological differences.

PCR analysis showing specific enrichment from low abundance target proteins
Figure 2: Normalization of biological differences in ChIP-Seq.

ChIP-Seq was performed on untreated cells and cells treated with a small molecule inhibitor of EZH2 methyltransferase. Using standard ChIP-Seq analysis (–) the differences in signal are not detected. Incorporation of the Spike-in Normalization Strategy (+) reveals the expected decrease in H3K27me3 ChIP-Seq signal confirming the value of the normalization strategy for detecting biological changes.


Specificity of Detection

The Spike-in chromatin consists of Drosophila melanogaster chromatin prepared from Schneider's Drosophila Line 2 (S2) cells. The Spike-in antibody recognizes a Drosophila-specific Histone variant, H2Av. Because of the specificity of the Spike-in antibody for the Spike-in chromatin modification, there is no cross-reactivity with mammalian samples leading to reduced background signal.

Specificity of the Spike-in Antibody
Figure 3: Specificy of the Spike-in Antibody.

The Spike-in antibody shows minimal cross reactivity with mammalian samples. When the Spike-in antibody was tested in ChIP-Seq with human chromatin, there is little to no signal detected. This demonstrates the specificity of the spike-in normalization strategy.

Contents & Storage

Please note that the ChIP Normalization reagents are available separately. Both the Spike-in Chromatin and Spike-in Antibody are required to apply the normalization strategy. Drosophila-specific qPCR primer sets are available for ChIP qPCR analysis.

Spike-in Antibody

  • 50 µg Spike-in Antibody supplied at a concentration of 1 µg/µl in PBS containing 0.035% sodium azide and 30% glycerol. Spike-in Antibody is shipped at room temperature. This will not affect the stability or performance of the reagent. Upon receipt, store at -20°C. Avoid repeated freeze/thaw cycles.

Spike-in Chromatin

  • Spike-in Chromatin prepared from Schneider's Drosophila Line 2 (S2) cells is provided for 15 rxns of robust histone modification antibody targets. Spike-in chromatin is provided at a concentration of 10 ng/µl.

qPCR Primer Sets

  • 400 µl Drosophila Positive Control Primer Set Pbgs is supplied at a concentration of 2.5 µM
  • 400 µl Drosophila Negative Control Primer Set 1 is supplied at a concentration of 2.5 µM