The EpiQuik ™ Chromatin Accessibility Assay Kit is a comprehensive set of optimized reagents designed to perform specific genetic analyses of chromatin accessibility, including nucleosome/transcription factor positioning of various biological samples using real-time PCR. It is a cost-effective and useful tool for the rapid detection of open and closed chromatin. The kit has the following advantages and features:
- The extremely fast and convenient protocol allows the entire procedure – from cell tissue sample to ready-to-use DNA for PCR – to be completed in as little as 1 hour and 30 minutes.
- It can be used with cultured cells and also with fresh and frozen tissues.
- The rapid process minimizes nuclear damage and the loss of dissociated chromatin components, preserving the chromatin structure.
- Human internal control primers are included in the kit as references to test the degree of chromatin accessibility to a specific target gene in the sample DNA, and also to validate whether proper enzymatic digestions are achieved.
- The option of a single reaction assay or high throughput multiple reaction assay can be used, making the assay flexible.
The accessibility of chromatin regulatory elements is critical to many aspects of gene regulation. Nucleosomes placed on regulatory elements inhibit the access of transcription factors to DNA. To elucidate the role of interactions between chromatin and transcription factors, it is crucial to determine chromatin accessibility by mapping nucleosome positioning throughout the genome.
In general, the more condensed the chromatin is, 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 the surrounding genes are to be actively transcribed. The presence (or 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 and procedure
The EpiQuik Chromatin Accessibility Assay Kit contains all the reagents necessary to obtain a specific genetic analysis of chromatin accessibility from cell/tissue samples using real-time PCR. This kit includes internal control primers to determine if chromatin digestion is successful. In this assay, chromatin is isolated from cells/tissues and treated with a mixture of nucleases.
The DNA is then isolated and amplified with real-time PCR for region-specific analysis of chromatin accessibility. Chromatin states can be identified based on the accessibility of DNA to nucleases. DNA in heterochromatin is inaccessible to foreign proteins, including exogenous nucleases, protecting it by nucleosome or DNA / protein complexes, and is available for subsequent PCR with negligible Ct changes between digested and undigested samples.
In contrast, DNA in euchromatin (nucleosome depletion) is accessible to exogenous nucleases, making it susceptible to nuclease digestion and becoming unavailable for PCR with a large Ct shift between digested and sampled samples. undigested.
Starting materials and input quantity
Starting materials can include various mammalian cell or tissue samples, such as flask or microplate culture cells, fresh and frozen tissues, etc. The amount of cells/tissues for each reaction can be from 1 x 10 5 cells or 2 mg of tissues to 2 x 10 6 cells or 40 mg of tissues. For an optimal reaction, the amount of input chromatin should be approximately 0.5 x 10 6 cells or 10 mg of tissues.