The EpiQ™ chromatin analysis kit developed by Bio-Rad Laboratories is a real-time PCR assay for the quantitative assessment of chromatin states in cultured cells. Using this kit, chromatin structure data can be obtained within six hours directly from cultured cells (a few as 5 x 104) without the need for nuclei isolation.
In situ 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 from nuclease digestion and available for subsequent quantitation by qPCR. Analysis of heterochromatin using the EpiQ kit reveals a minimal Cq shift between digested and undigested samples (Figure 2).
In contrast, the DNA in euchromatin is accessible to exogenous nucleases, making it susceptible to nuclease digestion and unavailable for qPCR. Analysis of euchromatin using the EpiQ kit shows a large Cq shift between digested and undigested samples (Figure 3).
By combining in situ chromatin digestion, genomic DNA purification, and real-time PCR, the chromatin state for several gene promoters can be studied simultaneously using the EpiQ chromatin analysis kit. The kit helps quantify the impact of epigenetic events, such as DNA methylation and histone modification, on gene-expression regulation through chromatin state changes.
The EpiQ chromatin analysis kit requires cultured (adherent or suspension) cells as starting material and includes the necessary supplies for performing chromatin assessment. Kit components include buffers for cell permeabilization and in situ chromatin digestion, optimized nuclease, materials for genomic DNA purification, control assays (qPCR primers) for chromatin assessment of a reference (epigenetically silenced) and control (constitutively expressed) gene, and the EpiQ chromatin SYBR® Green supermix, a real-time PCR reagent designed to amplify genomic DNA. Experimental results for user-specified gene targets are analyzed against the reference target to quantify the extent of chromatin accessibility.