ChIP-IT® FFPE II

FFPEサンプル用ChIPキット

ホルマリン固定パラフィン包埋(FFPE)試料は、遡及的な研究のための価値ある資源です。しかし、FFPE試料を用いた解析は,ChIPのような実験手法では使用できないことも少なくありませんでした。アクティブ・モティフ社は、これまでのクロマチン免疫沈降(ChIP)の専門知識を活用し、FFPE組織からのサンプル調製、ChIP、次世代シーケンシングまで可能とする最初の市販キットを開発しました。アクティブ・モティフ社では、研究の目的、出発試料や量に応じた2つのクロマチン調製キットと、ChIP実験のためのChIP-IT® FFPE II Kitをご用意しています。

FFPE ChIP-Seq results of glipblastoma patient-derived xenograft samples.
患者由来のグリオブラストーマ(膠芽腫)の異所移植サンプルからのFFPE ChIP-Seqの結果

(Click image to enlarge)

一般的にFFPE試料は,量に制限があるほか,ホルマリン固定法にも一貫性がなかったり,厳しい固定条件または長期保管のために,しばしば抗原性の劣化および喪失が観察されます。これらの要因はクロマチン調製を難しくし,また場合によっては最適化が必要とされます。ChIP-IT® FFPE Chromatin Preparation Kit は,固定が過剰だったり、保存期間が長く一般的に調製が難しいと思われる組織スライドまたは組織切片から高品質なクロマチンを抽出するために,特別に調製された試薬とプロトコールから構成されています。またChIP-IT® FFPE Chromatin Preparation II Kitは、少ない作業時間で、どちらかというと新鮮で高品質のFFPEサンプルからのクロマチン調製に適しています。

Choosing your FFPE Chromatin Prep Kit.

ChIP-IT® FFPE II Kit は、FFPE試料から得られたわずかなクロマチンから、ヒストンのように多量に含まれる対象物または転写因子のような少ない量のタンパク質の両方の濃縮を行うために必要な全ての試薬とプロトコールから構成されています。ChIP反応におけるプレクリアのステップは、バックグラウンドのシグナルを抑制し、より良いデータの取得に貢献しています。本キットには、ChIP実験の成功を評価するための、抗体およびPCRプライマーも提供されます。

ChIP-IT® FFPE II 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。

Active Motif also offers products compatible with ChIP-IT® FFPE II:

 
Name Format Cat No. Price  
ChIP-IT® FFPE Chromatin Preparation Kit 5 rxns 53030 ¥46,000 Buy
ChIP-IT® FFPE Chromatin Preparation Kit II 5 rxns 53031 ¥46,000 Buy
ChIP-IT® FFPE II 16 rxns 53047 ¥95,400 Buy

ChIP-IT® FFPE II Advantages

  • Obtain quality chromatin using histological slides or tissue blocks as the starting material
  • Sensitive enrichment of DNA from nanogram quantities of FFPE chromatin
  • Optimized reagents reduce background levels caused by non-specific binding events
  • Highly robust procedure has been validated using FFPE chromatin from both normal and tumor samples with proven performance in both qPCR and ChIP-Seq analysis
  • Controls are included in both the chromatin preparation and ChIP kits to help confirm results

 

How does the ChIP-IT® FFPE Chromatin Preparation Kit work?

Flow chart of ChIP-IT FFPE Chromatin Preparation Kit
Figure 1: Schematic of chromatin preparation from FFPE slides or sections.

In ChIP-IT® FFPE Chromatin Preparation, 10-20 µm sections from FFPE tissue blocks, or 5-10 µm sections from unstained FFPE slides are used as the starting material. Up to ten tubes or slides can be used for each chromatin preparation in order to obtain sufficient yield for downstream ChIP analysis. The paraffin is removed and the tissue is rehydrated from each slide/tube. The tissue then undergoes an incubation in lysis buffer, tissue homogenization, a 30 second sonication followed by digestion with Enzymatic Shearing Cocktail. After a centrifugation step, all remaining sample is pooled into a single tube for sonication. A final centrifugation is performed and the soluble and insoluble chromatin is analyzed and quantified by qPCR.

 

How does the ChIP-IT® FFPE Chromatin Preparation II Kit work?

Flow chart of ChIP-IT FFPE Chromatin Preparation Kit
Figure 2: Schematic of chromatin preparation from FFPE slides or sections.

In ChIP-IT® FFPE Chromatin Preparation II, 10 µm sections from FFPE tissue blocks, or 5-10 µm sections from unstained FFPE slides are used as the starting material. Up to five sections or slides can be used for each chromatin preparation in order to obtain sufficient yield for downstream ChIP analysis. The paraffin is removed and the tissue is rehydrated from each slide/tube. The tissue then undergoes a sonication. A final centrifugation is performed and the soluble and insoluble chromatin is analyzed and quantified by qPCR.

 

How does the ChIP-IT® FFPE II Kit work?

Flow chart of ChIP-IT FFPE II Kit
Figure 3: Schematic of chromatin immunoprecipitation using ChIP-IT FFPE II.

The ChIP-IT® FFPE II Kit requires chromatin prepared from FFPE slides or tissue blocks using the ChIP-IT® FFPE Chromatin Preparation or ChIP-IT® FFPE Chromatin Preparation II Kits (Catalog No. 53030/53031). Chromatin is pre-cleared followed by an incubation with an antibody directed against the DNA-binding protein of interest. The antibody-bound protein/DNA complexes are immunoprecipitated through the use of Protein G agarose beads and washed via gravity filtration. Following immunoprecipitation, cross-links are reversed, the proteins are removed by Proteinase K and the DNA is recovered and purified. ChIP enriched DNA can be used for either gene-specific or whole-genome analysis.

ChIP-IT® FFPE Chromatin Preparation

It can be difficult to extract chromatin from FFPE samples due to their crosslinked nature. Relatively low quality 10-year old kidney samples were processed using the ChIP-IT FFPE Chromatin Preparation Kit. To maximize the soluble chromatin available for ChIP enrichment, a subset of these samples underwent an additional round of sonication. Chromatin isolated from both rounds of sonication were combined to achieve enough input for the ChIP-IT FFPE Kit.

 

Solubility of chromatin after sonication

 

Solubility of chromatin following a second sonication
Figure 1: Analysis of chromatin solubility from human kidney FFPE samples.

Chromatin was extracted from two different case studies (A and B) from 10-year old histological sections of a human kidney with matched normal (N) and tumor (T) samples. Chromatin preparations were performed in duplicate. The soluble and insoluble chromatin fractions were analyzed by qPCR using the positive control GAPDH-2 primer set and DNA standards for quantification. The top image shows that only the tumor sample from case B had significant yields in the soluble fraction for use in downstream ChIP analysis. The insoluble pellets of the normal sample from case B and both normal and tumor samples from case A were diluted to 1 ml final volume with ChIP Buffer and subjected to a second round of sonication for 20 pulses at an amplitude of 63%. A 25 µl sample was collected from the new soluble and insoluble fractions and the input was reverse cross-linked, proteinase K treated and analyzed by qPCR using the GAPHD-2 primer set and DNA standards. Again, the solubility of the chromatin remained low. However, by combining the soluble fractions of the replicates, the chromatin yield was high enough to proceed with ChIP analysis.

ChIP-IT® FFPE Chromatin Preparation II

For high quality FFPE samples, like the glioblastoma patient-derived tumors shown below, the ChIP-IT FFPE Chromatin Preparation II Kit is an easy and quick protocol for isolation of chromatin. Following isolation, chromatin can be quantified by qPCR (as shown below) or Qubit.

qPCR Analysis of Glioblastoma Samples Assayed Using the ChIP-IT FFPE II Kit
Figure 2: qPCR Analysis of Glioblastoma Samples Assayed Using the ChIP-IT FFPE II Kit.

Samples of glioblastoma patient-derived xenografts were processed using the ChIP-IT FFPE Chromatin Preparation Kit. Three 5 µm curls were used for Sample GBM1, and cell pellets from dissociated tumor were used for Samples GBM2 and GBM3. Samples were reverse cross-linked, proteinase K treated and purified for analysis by qPCR using the included DNA standards and qPCR primer sets to determine the quantity and quality of the extracted chromatin. If sufficient quantities are not obtained after a single sonication, additional rounds of sonication can be performed.

ChIP-IT® FFPE II

The ChIP-IT® FFPE II Kit has the sensitivity required to work with extremely limited starting material, while producing minimal background signal, thereby enabling specific detection of the target protein of interest. The kit utilizes optimized reagents that preclear the chromatin samples and reduce background levels caused by non-specific binding events. Filtration based washes are fast and easy and result in improved consistency since there is no loss of sample material.

Due to the high sensitivity of the ChIP-IT FFPE II Kit, enriched chromatin can be used for both endpoint qPCR and NGS analysis. We recommend using the Next Gen DNA Library Kit to construct high complexity ChIP-Seq libraries.

H3K27ac and H3K27me3 ChIP-Seq on Glioblastoma Samples.
Figure 3: H3K27ac and H3K27me3 ChIP-Seq on Glioblastoma Samples..

The Samples of glioblastoma patient-derived xenografts were used in H3K27ac and H3K27me3 ChIP-Seq to generate genome-wide profiles of this histone modification. Libraries were constructed using the Next Gen DNA Library Kit (Catalog No. 53216).

Contents & Storage

ChIP-IT® FFPE Chromatin Preparation Kit

Please note that the ChIP-IT FFPE Chromatin Preparation Kit is delivered using two separate shipping temperatures, a room temperature and a dry ice shipment. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Please confirm receipt of all reagents upon arrival and store items at the appropriate temperatures as listed below:

  • RNaseA (10 mg/ml); Store at -20°C
  • Proteinase K (10 mg/ml); Store at -20°C
  • Protease Inhibitor Cocktail; Store at -20°C
  • Precipitation Buffer; Store at -20°C
  • Carrier; Store at -20°C
  • Enzymatic Shearing Cocktail; Store at -20°C
  • DNA Standard AM1; Store at -20°C
  • DNA Standard AM2; Store at -20°C
  • DNA Standard AM3; Store at -20°C
  • Human Positive Control Primer Set GAPDH-2; Store at -20°C
  • Lysis Buffer AM5; Store at 4°C
  • Digestion Buffer AM2; Store at 4°C
  • Paraffin Removal Solution; Store at RT
  • ChIP Buffer; Store at RT
  • 5 M NaCl; Store at RT
  • TE pH 8.0; Store at RT
  • DNA Purification Elution Buffer; Store at RT

ChIP-IT® FFPE Chromatin Preparation II Kit

Please note that the ChIP-IT FFPE Chromatin Preparation II Kit is delivered using two separate shipping temperatures, a room temperature and a dry ice shipment. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Please confirm receipt of all reagents upon arrival and store items at the appropriate temperatures as listed below:

  • RNaseA (10 mg/ml); Store at -20°C
  • Proteinase K (10 mg/ml); Store at -20°C
  • Protease Inhibitor Cocktail; Store at -20°C
  • Deacetylase Inhibitor; Store at -20°C
  • Carrier; Store at -20°C
  • PMSF; Store at -20°C
  • DNA Standard AM1; Store at -20°C
  • DNA Standard AM2; Store at -20°C
  • DNA Standard AM3; Store at -20°C
  • Human Positive Control Primer Set GAPDH-2; Store at -20°C
  • FFPE ChIP Buffer; Store at RT
  • 5 M NaCl; Store at RT
  • TE pH 8.0; Store at RT
  • FFPE Low EDTA TE; Store at RT 

ChIP-IT® FFPE II Kit

The ChIP-IT FFPE Kits are shipped on dry ice and contain reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. Do not re-freeze the Protein G Agarose Beads after you have received this kit. This kit includes the following components:

  • Proteinase K (10 mg/ml); Store at -20°C
  • 10X PBS; Store at -20°C
  • 100 mM PMSF; Store at -20°C
  • Protease Inhibitor Cocktail; Store at -20°C
  • Carrier; Store at -20°C
  • Blocker; Store at -20°C
  • Blocking Reagent AM1; Store at -20°C
  • BSA (10 mg/ml); Store at -20°C
  • Protein G Agarose Beads; Store at 4°C
  • Histone H3K9ac rAb; Store at -20°C
  • Human Positive Control Primer Set GAPDH-2; Store at -20°C
  • Human Negative Control Primer Set 2; Store at -20°C
  • ChIP Buffer; Store at RT
  • ChIP Filtration Columns; Store at RT
  • 5 M NaCl; Store at RT
  • TE pH 8.0; Store at RT
  • Wash Buffer AM1; Store at RT
  • LiCl Buffer; Store at RT
  • Elution Buffer AM4; Store at RT
  • 1.7 ml siliconized tubes; Store at RT
  • Low EDTA TE; Store at RT