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Jun 1, 2011 (Vol. 31, No. 11)

PCR Assay for Chromatin Accessibility

Epigenetics Tool for the Rapid Determination of Chromatin State for Gene-Expression Studies

  • Epigenetics Analysis

    Click Image To Enlarge +
    Figure 2. The DNA in heterochromatin is inaccessible to exogenous nuclease and is available for qPCR. The result is a minimal Cq shift between digested and undigested samples.

    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).

  • Click Image To Enlarge +
    Figure 3. The DNA in euchromatin is accessible to exogenous nuclease and is unavailable for qPCR. The result is a large Cq shift between digested and undigested samples.

    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.

  • Making Connections in Cancer Cell Proliferation

    Together with a group of Japanese researchers, Ian Marc Bonapace, Ph.D., discovered the UHRF1 protein in the late 1990s. Since its discovery, UHRF1 has been shown to play an important role in gene regulation; specifically, it controls the epigenetic machinery that reinstalls correct modifications on DNA and chromatin during cell proliferation.

    The main objective of Dr. Bonapace’s studies is to determine ways UHRF1 can be used to improve cancer treatment. “We believe that, at minimum, this protein can be very useful as a marker for cancer aggressiveness,” explains Bonapace. “But we hope to discover in the future that hampering the activity of UHRF1 could be a way to stop tumor growth and metastasis in the body.”

    Early in 2010, Dr. Bonapace obtained the EpiQ kit. To test its efficacy and applicability to his research efforts, he conducted an experiment to compare the expression of three different oncosupressors in two different lines of prostate cancer.

    Dr. Bonapace explains that prior to obtaining the EpiQ kit, determining if genes are expressed or not required days of work using the more resource-intensive chromatin immunoprecipitation method. “With this kit,” he explains, “you can determine immediately if genes are repressed or not. Then you know whether or not to go through the more time-consuming and money-consuming experiments to study why.”



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