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?
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?
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.
||Sample used per chromatin preparation
||Tissue block – five 20 µm sections
||Tissue block – twenty-five 20 µm sections
||Tissue block – two 20 µm sections
|Rat whole brain
||5 slides – two 5 µm sections
||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.
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.
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.