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ChIP-IT® FFPE

perform ChIP from FFPE samples

Formalin-fixed paraffin-embedded (FFPE) clinical samples are a valuable resource for retrospective research. However, working with FFPE samples presents a challenge for techniques such as ChIP. Active Motif has leveraged its ChIP expertise to develop the first commercially available kits to enable ChIP from FFPE tissue for use in Next Generation sequencing. The FFPE ChIP assay comes in two modules, the ChIP-IT® FFPE Chromatin Preparation Kit and ChIP-IT® FFPE Kit, to allow flexibility dependent on type and amount of starting material.

FFPE ChIP-Seq results of normal and tumor human colon samples.
FFPE ChIP-Seq results of normal and tumor human colon samples.

(Click image to enlarge)

FFPE samples are typically limited in size, lack consistency in the methods used for formalin fixation and often display degradation and loss of antigenicity due to harsh fixation conditions or prolonged storage. These factors can affect the ease of chromatin preparation and may require a certain degree of optimization. The ChIP-IT® FFPE Chromatin Preparation Kit contains specially formulated reagents and protocol guidelines to extract high quality chromatin from histological slides or tissue sections.

The ChIP-IT® FFPE Kit contains all the necessary reagents and protocols to allow enrichment of both high abundance targets, such as histones, or low abundance proteins, such as transcription factors from the limited amounts of chromatin obtained from FFPE samples. Antibody and PCR primer controls are also provided in this kit to evaluate the successful execution of the ChIP procedure.

To learn more about ChIP-IT® FFPE, click on the Method, Data, or Contents tabs below. To view a manual or other related documents, click on the Documents tab below.

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

 
Name Format Cat No. Price  
ChIP-IT® FFPE Chromatin Preparation Kit 5 rxns 53030 $235 Buy Now
ChIP-IT® FFPE 16 rxns 53045 $495 Buy Now

ChIP-IT® FFPE 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
  • Filtration based washes offer a faster, easier solution with better consistency than magnetic capture
  • 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 Kit work?

Flow chart of ChIP-IT FFPE Kit
Figure 2: Schematic of chromatin immunoprecipitation using ChIP-IT FFPE.

The ChIP-IT® FFPE Kit requires chromatin prepared from FFPE slides or tissue blocks using the ChIP-IT® FFPE Chromatin Preparation Kit (Catalog No. 53030). Chromatin is then incubated 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

The detection limits of the assay will depend on the size and type of FFPE tissue being used. Due to the variability in the formalin fixation process and the storage conditions of the FFPE sample, not all FFPE material may yield high quality chromatin. Active Motif's protocol offers guidelines and troubleshooting tips for processing samples to obtain the required minimum of 200 ng chromatin per ChIP reaction. The table below shows examples of FFPE samples and the number of tissue slides or sections used per chromatin preparation that have been successfully used in the ChIP-IT® FFPE.

Tissue type Sample used per chromatin preparation
Human Colon Tissue block – five 20 µm sections
Human Kidney Tissue block – twenty-five 20 µm sections
Human Lung Tissue block – two 20 µm sections
Rat whole brain 5 slides – two 5 µm sections
Rat hippocampus 25 slides – two 5 µm sections

 

Due to the limited sample size of FFPE material, it is often necessary to process multiple slides or tissue sections together during a single chromatin preparation to obtain enough chromatin for downstream ChIP analysis. Enough reagents are included in the ChIP-IT® FFPE Chromatin Preparation Kit to process 10 slides or tubes per preparation. Each FFPE sample may perform differently during the chromatin preparation. Even the same tissue type may have a different level of difficulty during the tissue homogenization and sonication depending on the formalin fixation process used or if the tissue is from a normal or tumor sample.

The ChIP-IT® FFPE Chromatin Preparation Kit includes DNA standards and PCR primers for the quantification of chromatin by qPCR analysis. Due to the low concentration of the Input material from FFPE chromatin extractions, we do not recommend analysis of the shearing efficiency by agarose gel electrophoresis. Instead, we recommend evaluating the quantity and quality of the chromatin preparation by qPCR. The ChIP-IT® FFPE Chromatin Preparation Kit includes DNA standards and PCR primers which can be used to quantify the chromatin and verify if the chromatin has been efficiently solubilized for use in downstream ChIP analysis. If necessary, the chromatin can undergo additional rounds of sonication to improve the solubility and overall yield.

Solubility of chromatin after sonication

 

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

The ChIP-IT® FFPE Kit requires chromatin prepared from FFPE slides or tissue blocks using the 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

Once sufficient chromatin is available in the soluble fraction to perform the desired ChIP reactions including replicates and controls, it is time to proceed with the ChIP-IT® FFPE Kit. The ChIP-IT® FFPE Kit is the only ChIP Kit available that 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 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.

qPCR results of FFPE human colon with H3K4me3 antibody

 

qPCR results of FFPE human colon with YY1 antibody
Figure 2: qPCR analysis of normal and tumor human colon samples assayed using ChIP-IT FFPE.

The ChIP-IT® FFPE Kit requires chromatin prepared from FFPE slides or tissue blocks using the Chromatin was extracted from 10-year old matched normal and tumor FFPE tissue block sections of a human colon using the ChIP-IT FFPE Chromatin Preparation Kit. 250ng and 185 ng of normal and tumor chromatin was used per ChIP reaction in the ChIP-IT FFPE Kit. Antibodies for histone H3K4me3 or transcription factor YY1 were used for enrichment according to the recommendations in the manual. The quality of the ChIP-enriched DNA was then validated using the ChIP-IT qPCR Analysis Kit (Catalog No. 53029), which enables normalization of the data to account for differences in chromatin amounts, primer efficiency and ChIP elution volumes. The qPCR results for each ChIP antibody are shown above. The H3K4me3 results match observed data showing that GAPDH is up-regulated in certain cancers. The data represents triplicate values expressed as Binding events detected per 1,000 cells.

 

Due to the high sensitivity of the ChIP-IT FFPE® Kit, sequencing libraries can be generated from sub-nanogram levels of ChIP DNA. For good ChIP-Seq data, quality enrichments with low background is more important than the total quantity of DNA recovered. A typical ChIP-Seq reaction will yield 10-20 million unique alignments, but due to the limited size of FFPE samples, the number of unique alignments will be reduced to a range of 2-10 million. Although these results are not as robust as a typical ChIP-Seq data set, these data sets can still be reliably interpreted.

qPCR results of FFPE human colon with YY1 antibody
Figure 3: ChIP-IT® FFPE ChIP-Seq results of normal and tumor human colon samples.

The H3K4me3 ChIP-Seq results from tumor and normal FFPE colon samples and YY1 ChIP-Seq from normal colon are displayed above. Only the portion of the genome-wide data surrounding the YY1 gene is shown. The top panel shows YY1 binding at the promoter of the YY1 gene, thus illustrating the known autoregulation of YY1. The middle and bottom sequence represent H3K4me3 binding the YY1 gene in normal and tumor, respectively, indicating YY1 is expressed in both the normal and tumor samples.

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 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:

  • Protein G Agarose beads; Store at 4°C
  • Proteinase K (10 µg/µl); Store at -20°C
  • Blocker; Store at -20°C
  • Protease Inhibitor Cocktail (PIC); Store at -20°C
  • Histone H3K4me3 pAb; 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
  • TE, pH 8.0; Store at RT
  • ChIP Filtration Columns; Store at RT
  • ChIP Buffer; Store at RT
  • Wash Buffer AM1; Store at RT
  • Elution Buffer AM4; Store at RT
  • DNA Purification Binding Buffer; Store at RT
  • 3 M Sodium Acetate; Store at RT
  • DNA Purification Wash Buffer; Store at RT
  • DNA Purification Elution Buffer; Store at RT
  • DNA Purification Columns; Store at RT