Posts Tagged ‘EpiQ chromatin analysis kit’

Stem cell research and other bed time stories

 :: Posted by American Biotechnologist on 10-05-2011

Dr David Schaffer

Bedtime stories in Dr David Schaffer’s childhood home were often not standard fairy tales. With both parents in careers as biomedical researchers (his mother in drug development and clinical trials for a major pharmaceutical company and his father, in cardiovascular research and a pharmacology professor) much of the conversation as far back as Schaffer can remember centered on biology and science. “I remember being five years old and sitting on my father’s lap, while he was teaching me the names of microorganisms,” says Schaffer. In many ways, a life dedicated to solving biological problems seemed predetermined, but Schaffer did deviate slightly from the examples and influences of his parents and initially concentrated on the engineering aspects of the field.

Schaffer completed his undergraduate work at Stanford University in chemical engineering and graduate work in chemical engineering at MIT. His postdoctoral work was in the laboratory of Dr Fred Gage, a neurobiologist at the Salk Institute for Biological Studies. “For two years, I was the only engineer at the Salk Institute, and had immersed myself in the rich world of biology in a lab that had been making some paradigm-shifting discoveries in the field of neural stem cells and understanding how the adult brain continues to add neurons,” says Schaffer. It was during this pivotal period that Schaffer became fascinated with applying engineering approaches to the study of problems in stem cell biology.

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PCR Assay for Chromatin Accessibility

 :: Posted by American Biotechnologist on 06-10-2011

Epigenetics is the study of inherited phenotypic changes caused by mechanisms other than mutations in the underlying DNA sequence. In mammalian cells, most of the chromatin—a complex of DNA, proteins, and histones—exists in a condensed, transcriptionally silent form called heterochromatin. The transcriptionally active form of chromatin is called euchromatin; it exists in a relaxed, less condensed state.

Histone subunits and DNA can be chemically modified as a result of environmental factors. These chemical modifications, called epigenetic markers (or marks), which include DNA methylation and histone tail modification (Figure 1), influence chromatin structure by altering its electrostatic nature or by modulating the affinity of chromatin-binding proteins. By altering chromatin structure, epigenetic changes have a profound effect on the expression of the genes present in the genomic regions affected.

Histones are strongly alkaline proteins that package and order DNA into structural units called nucleosomes. They are the major protein component of chromatin and can undergo several covalent chemical modifications, including methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation. A clear correlation has been established between histone acetylation and active transcription. Conversely, many histone methylation events are correlated with transcriptional silencing. Different histone modifications likely function in different ways; acetylation at one position will have a different effect than acetylation at another position.

Multiple modifications exist simultaneously and likely work together to influence chromatin state and gene expression. The concept of multiple dynamic modifications regulating gene expression in a systematic and reproducible fashion is known as the histone code.

In eukaryotes, DNA can be modified by methylation of cytosine bases. The enzymes that carry out this modification are called DNA methyltransferases. Aberrant or increased levels of methylation has been correlated with gene silencing and the development of several cancers. Recently, a second cytosine modification, 5-hydroxymethylcytosine (5-hmC), has been characterized in eukaryotes and research efforts are focused to understand its function.

Methods Used to Study Epigenetics
The most common technique for assessing DNA methylation involves the use of sodium bisulfite to convert unmethylated and methylated cytosine residues to uracil and cytosine, respectively. Methylated cytosines can then be identified through various downstream nucleic acid analysis methods, including PCR, qPCR, HRM, and sequencing. This commonly used technique can both identify individual methylation sites and quantify the level of methylation for a particular genomic region.

Other techniques for methylation analysis include the use of restriction enzymes, which are either resistant or sensitive to DNA methylation, and methylated DNA immunoprecipitation (MeDIP), which utilizes antibodies to isolate methylated DNA fragments for analysis.

The interaction between proteins (for example, histones) and DNA is most often studied using a technique known as chromatin immunoprecipitation (ChIP). ChIP is a preparatory method that uses highly specific antibodies against DNA-binding proteins to isolate DNA fragments that bind transcription factors or other DNA-binding proteins.

The isolated fragment can be characterized by using various nucleic acid analysis techniques, including PCR, qPCR, sequencing, and microarray hybridization. This can help determine whether specific proteins are associated with specific genomic regions. ChIP is also useful for identifying regions of the genome that are associated with specific histone modifications (for example, activating or repressive).

Researchers are interested in understanding the role of epigenetic changes, including DNA methylation and histone modifications, in disease (cancer) development and cell differentiation and reprogramming. The methods described thus far provide detailed molecular information about epigenetic markers that can be correlated to changes in gene-expression levels. However, they do not provide direct information regarding the chromatin state associated with these epigenetic marks.

Furthermore, several of these methods are time-consuming and may require up to five days to complete. New tools that deliver additional information about the functional state of chromatin in a shorter time are needed to accelerate the pace of epigenetics research.

Epigenetics Analysis
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.

Thanks to Viresh Patel, Ph.D. of Bio-Rad Laboratories for the guest post.

The First Epigenetics Tool that Quantifies Chromatin State for Gene Expression Studies

 :: Posted by American Biotechnologist on 02-15-2011

Bio-Rad Laboratories, Inc.’s new EpiQ chromatin analysis kit is a real-time PCR assay for the rapid quantitative assessment of chromatin structure. Complementing existing epigenetic assays such as DNA methylation and chromatin immunoprecipitation, the EpiQ kit is the first commercial research tool that helps scientists quantify the impact of epigenetic events on gene expression regulation through chromatin state changes.

“The EpiQ chromatin analysis kit provides researchers with an easy and fast way to quantify a gene’s chromatin state,” said Viresh Patel, Bio-Rad Marketing Manager for PCR reagents. “Using the kit, cancer, developmental, and stem cell biology researchers are able to glean direct information regarding the chromatin state that’s associated with epigenetic marks such as DNA methylation and histone modifications for the first time.”

Epigenetic processes such as DNA methylation and histone modification control gene expression by altering chromatin structure. Genes that are actively transcribed are associated with “accessible” chromatin regions called euchromatin, while genes that are transcriptionally silent are often in “inaccessible” chromatin regions called heterochromatin. The EpiQ kit can provide quantitative information about chromatin accessibility, which correlates very strongly with gene expression.

Reduces Analysis Time from Days to Hours
Until now, the only way to quantitatively probe the status of chromatin structure was through the use of homebrew methods, first described in 2001 (Rao, et. al., J. Immunol.). This approach requires the isolation and purification of nuclei from several millions cells prior to chromatin digestion and takes more than two days to complete. With the EpiQ kit, chromatin structure data can be obtained within six hours from as few as 50,000 cultured cells, without the need for nuclei isolation.

Key benefits of the EpiQ chromatin analysis kit include:

  • Assessment of chromatin structure in cultured cells within six hours
  • Requirement of relatively few cells (as few as 50,000) to perform analysis
  • Generation of quantitative chromatin structure information for multiple genomic elements

How It Works
The Chromatin state of a gene can be identified in situ based on its sensitivity to the action of the nuclease in the EpiQ kit. In heterochromatin, genomic DNA is inaccessible to nuclease digestion and remains available for subsequent qPCR. Analysis of heterochromatin using the EpiQ kit reveals a minimal quantification cycle (Cq) shift between digested and undigested samples. In contrast, DNA in euchromatin is susceptible to nuclease digestion and is unavailable for qPCR. Analysis of euchromatin using the EpiQ kit shows a large Cq shift between digested and undigested samples.

What Is Included in the EpiQ Kit
EpiQ 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 EpiQ™ Chromatin SYBR® Green Supermix, a real-time PCR reagent designed to amplify genomic DNA. The supermix can be used on all Bio-Rad real-time PCR instruments and on those from other suppliers; some instruments may require slight modifications to the supermix.

For more information about the EpiQ chromatin analysis kit, visit www.bio-rad.com/epiq.

For a layman’s guide to epigenetics see epigenetics for friends and family.