The EpiQuik™ Chromatin Accessibility Assay Kit is a complete set of optimized reagents designed for conducting a gene-specific analysis of chromatin accessibility including nucleosome/transcription factor positioning from various biological samples via real time PCR. It is a cost-effective and useful tool for quick detection of open and closed chromatin. The kit has the following advantages and features:
- Extremely fast and convenient protocol that allows the entire procedure (from cell tissue sample to ready-to-use DNA for PCR) to be finished in as short as 1 hour and 30 minutes.
- Able to be used with cultured cells and also fresh and frozen tissues.
- Fast process minimizes nuclear damage and loss of disassociated chromatin components, preserving chromatin structure.
- Internal control primers for human are included in the kit as references for analyzing the extent of chromatin accessibility in a specified gene target in the sample DNA, and also for validating whether the proper enzymatic digestions are achieved.
- Choice of single-reaction assay or high throughput multi-reaction assay can be used, making the assay flexible.
The accessibility of regulatory elements in chromatin is critical for many aspects of gene regulation. Nucleosomes positioned over regulatory elements inhibit access of transcription factors to DNA. To elucidate the role of the interactions between chromatin and transcription factors, it is crucial to determine chromatin accessibility through mapping of the nucleosome positioning along the genome. In general, the more condensed the chromatin, the more difficult it is for transcription factors and other DNA binding proteins to access DNA and carry out their tasks. The more accessible the DNA, the more likely surrounding genes are actively transcribed. The presence (or the absence) of nucleosomes directly or indirectly affects a variety of other cellular and metabolic processes such as recombination, replication, centromere formation, and DNA repair.
Principle & Procedure
The EpiQuik™ Chromatin Accessibility Assay Kit contains all the necessary reagents required for obtaining a gene-specific analysis of chromatin accessibility from cell/tissue samples via real time PCR. This kit includes internal control primers for determining whether chromatin digestion is successfully achieved. In this assay, chromatin is isolated from the cells/tissues and is treated with a nuclease mix. DNA is then isolated and amplified with real time PCR for region-specific analysis of chromatin accessibility. Chromatin states can be identified based on how accessible the DNA is to nucleases. The DNA in heterochromatin is inaccessible to outside proteins, including exogenous nucleases, rendering it protected by nucleosome or DNA/protein complexes and becomes available for subsequent PCR with insignificant Ct shifts between digested and undigested samples. In contrast, the DNA in euchromatin (nucleosome depletion) is accessible to exogenous nucleases, making it susceptible to nuclease digestion and becomes unavailable for PCR with a large Ct shift between digested and undigested samples.
Starting Materials & Input Amount
Starting materials can include various mammalian tissue or cell samples such as cells from flask or microplate cultured cells, fresh and frozen tissues, etc. The amount of cell/tissues for each reaction can be from 1 x 105 cells or 2 mg tissues to 2 x 106 cells or 40 mg tissues. For an optimal reaction, the input chromatin amount should be about 0.5 x 106 cells or 10 mg tissues.
Fig. 1. Schematic procedure of the EpiQuik™ Chromatin Accessibility Assay Kit.
Fig. 2. Chromatin accessibility of opened chromatin (euchromatin). Amplification of proximal promoter regions for the constitutively expressed target gene was carried out in MDA-231 cells by using positive control primers and using the EpiQuik™ Chromatin Accessibility Assay Kit (Cat. #P-1047) and EpiQuik™ Quantitative PCR Fast Kit (Cat. #P-1029). Red and green lines: Nse-untreated; Blue lines: Nse-treated. The difference of Ct values between Nse-treated and untreated is > 7 cycles.
Fig. 3. Chromatin accessibility of closed chromatin (heterochromatin). Amplification of proximal promoter regions for the constitutively repressed target gene was carried out in MDA-231 cells by using negative control primers and using the EpiQuik™ Chromatin Accessibility Assay Kit (Cat #P-1047) and EpiQuik™ Quantitative PCR Fast Kit (Cat #P-1029). Red and green lines: Nse-untreated; Blue and pink lines: Nse-treated. The difference of Ct values between Nse-treated and untreated is <1.5 cycles.