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ChIP-IT® qPCR Analysis Kit

ChIP DNAのqPCRデータ分析を単純化します

The ChIP-IT® qPCR Analysis Kit は,ChIP qPCR解析とデータ解釈を単純化するようにデザインされています。キットは,検量線用DNA,検量線用プライマーセット,ヒトおよびマウス用のポジティブおよびネガティブコントロールqPCRプライマーセットおよびデータを自動的に正規化してグラフで示す分析用スプレッドシートを含みます。正規化計算は,複数の実験で多くの試料の信頼性のある比較を可能にするデータ出力において,一貫性のレベルを取り込むために,ChIP反応で用いられたクロマチン量,ChIP DNAの再懸濁量およびプライマー効率を考慮に入れます。データ出力の一貫性に基づいて,研究者がChIP DNAの品質を確実に評価することができるように,アクティブ・モティフ社はデータ解釈に関して具体的な提言を行うことができます。アクティブ・モティフ社では,クロマチン免疫沈降反応を行うために,ChIP-IT High Sensitivity® Kit の使用を推奨します。

 ChIP-IT® qPCR Analysis Spreadsheet のコピーをダウンロードするためには,リンクをクリックしてください。

 
Name Format Cat No. 価格 (税抜)  
ChIP-IT® qPCR Analysis Kit 10 rxns 53029 ¥21,000 Buy Now

ChIP-IT® qPCR Analysis Kit Advantages

  • Simplifies qPCR data analysis to enable a faster, more reliable interpretation of data
  • All calculations are included in a simple Excel sheet in which data from your qPCR machine can be entered directly resulting in automatic data normalization and graph generation
  • Normalized data can be compared across multiple sample types and experiments
  • Includes human and mouse positive control and negative control qPCR primer sets
  • Recommendations for acceptable data values allows users to evaluate the quality of the ChIP reaction before proceeding with downstream applications such as ChIP-Seq

How does it work?

Using the ChIP DNA obtained from the ChIP-IT High Sensitivity Kit, qPCR reactions are set up for the provided DNA Standards, the ChIP DNA samples and Input DNA. An example 96-well plate qPCR set up using six different ChIP DNA samples and four different PCR primer pairs is shown below. Each ChIP and Input reaction is tested in triplicate using the provided species-specific negative control primer set and a positive control primer. Since the DNA mass in each standard is known, the number of copies of the genome present in each standard can be calculated (assuming that each cell contains 6.6 pg of DNA). The primer efficiency is calculated for each primer set and the qPCR values for the ChIP reactions are normalized against primer efficiency, the amount of chromatin used in the ChIP reaction and the resuspension volume of the ChIP DNA. To see a complete explanation of the data calculations and the recommendations for data interpretation, please download a copy of the product manual under the Documents tab.

qPCR plate set up for the ChIP-IT qPCR analysis kit
Figure 1: qPCR plate set up for use with the ChIP-IT qPCR Analysis Kit.

This example 96-well plate set up shows how the ChIP-IT qPCR Analysis Kit can be used to compare six different ChIP DNA samples with 4 different qPCR primer sets. Only one standard curve is needed for the entire plate.

Data Interpretation

Active Motif has done an extensive evaluation of ChIP DNA quality using the ChIP-IT qPCR Analysis Kit and normalization strategy. Based on these results we are able to provide specific recommendations for assessing ChIP DNA quality. These recommendations are based on the use of the ChIP-IT High Sensitivity Kit (Catalog No. 53040) to perform the chromatin immunoprecipitation reactions. If another method was used to perform the ChIP reactions, the interpretation results listed below may not apply. Below are a few example recommendations. A complete description can be found in the product manual under the Documents tab.

  • Negative control primers should give values of less than 2 Binding events detected per 1,000 cells. Values higher than 2 could indicate a poor quality ChIP reaction
  • ChIP reactions using antibodies against high abundance targets, such as histone modifications, will routinely give values between 100 and 1,000 Binding events detected per 1,000 cells.
  • ChIP reactions using antibodies against lower abundance targets, such as transcription factors, will routinely give values between 10 and 100 Binding events detected per 1,000 cells.
  • ChIP reactions that give values less than 5 Binding events detected per 1,000 cells are usually not of high quality. The ChIP reaction should be repeated or a different antibody should be used.
  • A successful ChIP reaction will have a minimum of 5-fold higher values with the positive control primers as compared to the negative control primers.
Example data analysis of good and poor ChIP qPCR results
Figure 1: The ChIP-IT qPCR Analysis Kit and normalization strategy provide an effective means of evaluating ChIP DNA quality.

The ChIP-IT qPCR Analysis Kit manual provides recommendations for evaluating the quality of ChIP reactions. The top image shows two examples of ChIP reactions that do not meet the recommended criteria for quality ChIP DNA. ChIP DNA from Cell Line A shows high background and less than 5-fold enrichment, while ChIP DNA from Cell Line B shows low signal strength. The bottom image show ChIP DNA that is considered high quality as both samples show low background and greater than 5-fold enrichment.

Example data analysis of the effect of normalization
Figure 2: The ChIP-IT qPCR Analysis Kit enables direct comparison of ChIP efficiency.

ChIP was performed using an RNA pol II antibody with the ChIP-IT High Sensitivity Kit. Different amounts of chromatin were used in each ChIP reaction ranging from 3,000 cell equivalents to 150,000 cell equivalents. In the top image normalization was performed independent of starting chromatin amount, taking only primer efficiency and resuspension volume into account. In the bottom image, using Active Motif's ChIP-IT qPCR Analysis Kit, starting amount of chromatin is accounted for. This data demonstrates the dynamic range of the ChIP-IT High Sensitivity Kit and the accuracy of the normalization strategy in the ChIP-IT qPCR Analysis Kit.

Contents & Storage

The ChIP-IT® qPCR Analysis Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below.

  • DNA Standard AM1; Store at -20°C
  • DNA Standard AM2; Store at -20°C
  • DNA Standard AM3; Store at -20°C
  • Standard Curve Primer Pair; Store at -20°C
  • Human Negative Control Primer Set 1; Store at -20°C
  • Human Positive Control Primer Set GAPDH-2; Store at -20°C
  • Mouse Negative Control Primer Set 1; Store at -20°C
  • Mouse Positive Control Primer Set Gapdh-2; Store at -20°C
  • 2 mM Tris-HCl, pH 8.0; Store at RT

ChIP qPCR data that has been normalized according to the ChIP-IT® qPCR Analysis Kit method has been published many times. Some select publications are listed below.