Gene expression studies are a mainstay in the search for blockbuster drugs and novel biomarkers. Scientists in recent years have turned to microarrays - small pieces of glass carefully arrayed and printed with microscopic spots of DNA. Use of microarrays is expanding beyond gene expression applications to address more complex issues such as chromosomal additions and deletions, introns and exons, gene regulatory sequences, SNPs, and translation modifiers such as microRNAs.
These and other points of molecular complexity build upon one another, allowing researchers to characterize pathways or networks to answer specific biological questions. One such application uses ChIP-on-chip technology.
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| The Developing Science of ChIP-on-chip Technology |
Where the proteome meets the genome
A new, highly-focused microarray application for gene expression is emerging that goes beyond traditional analyses. Traditional gene expression methods provide only a partial glimpse into gene regulation, but transcription is a complex process that requires multiple interactions and the orchestrated binding of numerous components.
This new platform explores transcriptional activities - both activation and repression - by allowing the determination of the precise location on the genomic DNA sequence where a regulatory protein is bound.
Agilent's ChIP-on-chip (chromatin immunoprecipitation-on-chip) is the powerful technology driving this next generation microarray platform. ChIP-on-chip, also known as Location Analysis (LA), provides insight into key mechanisms of methylation, histone modification, as well as DNA replication, modification, and repair. It has been used to understand diseases such as diabetes, leukemia, and breast cancer, and has already provided important insight to vital processes like cell proliferation, cell fate determination, oncogenesis, cell cycle, apoptosis, and neurogenesis.
ChIP-on-chip pairs chromatin immunoprecipitation (ChIP) with glass slide microarrays (chip) to analyze how regulatory proteins interact with the genome of living cells. Regulatory proteins bind to genomic DNA to control chromosome replication and gene activity, thereby functioning as switches in the regulatory circuitry of cells. This network of circuits is uncharted in many instances and its understanding will aid researchers and companies in identifying new target genes and therapeutics capable of modulating these pathways. Combining the new information gained from ChIP-on-chip studies with the wealth of already available gene expression data will help speed and focus both disease research and drug discovery.
This technology has been extensively developed by the Computational Biology Corporation, a biotech pioneer in ChIP-on-chip that was founded by Drs. Richard Young and David Gifford of the Massachusetts Institute Technology and the Whitehead institute for Biomedical Research, as well as biotechnology executive Dr. Heidi Wyle. This company was acquired by Agilent in January of 2005, giving Agilent exclusive access to patent and intellectual property for ChIP-on-chip analysis. The acquisition allows Agilent to provide unique microarray solutions for disease research, drug discovery, and drug development.
The most recent compelling validation of ChIP-on-chip was done by researchers at the Whitehead Institute laboratory of Dr. Richard Young. They used ChIP-on-chip to analyze how human embryonic stem cells retain their unique ability to differentiate into any cell type as well as what underlying processes strip away this pluripotency once the cells move along specific differentiation pathways. Results of these efforts have been published in the Sept. 23, 2005 issue of the journal Cell (Vol. 122 (6), 947-956). Additionally, ChIP-on-chip has been skillfully applied in many other ways to answer key questions about the role of oncoproteins in cell cycle progression, apoptosis, and myogenesis.
By partnering our unparalleled microarray expertise with exclusively patented technology from the Whitehead institute, Agilent enables you to obtain insightful and robust profile of DNA-protein binding and promoter occupancy across entire genomes. Acquire more complete and reliable information for networks of in vivo gene expression in specific cells, tissues, or entire organisms in a matter of weeks - information that previously would have taken years.
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| Applications Enabled by the Agilent ChIP-on-chip System |
ChIP-on-chip technology enables the researcher to successfully focus on many critical areas previously hampered by the lack of useful tools to complete the studies. It allows researchers to:
- Uncover and validate gene regulation and regulatory networks by comprehensive determination of promoter occupancy.
- Identify and characterize molecular events associated with processes for transcription, DNA replication and repair, as well as with chromatin modifications and DNA methylation.
- Elucidate modes of action and potential therapeutic activities of compounds and target genes by mapping gene regulatory networks relevant to disease and pathophysiological states.
- Validate and augment existing gene expression data with authentic binding events.
- Identify, assess, and monitor biomarkers response to protein-DNA binding events to serve as bioassays or toxicant signatures for toxicogenomics.
- Uncover and profile off-target events as well as validate primary and secondary effects in screening of candidate compounds, siRNAs, therapeutics, etc.
Featured Publication
Control of developmental regulators by Polycomb in human embryonic stem cells
Lee et. al, (2006) Cell 125(2):301-13
Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. The Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells was mapped genome-wide using Agilent ChIP-on-chip microarrays. SUZ12 was found to be distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. The researchers also found that PRC2 target genes are preferentially activated during ES cell differentiation. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation. 
Additional publications describing ChIP-on-chip technology
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| Key Features and Benefits of the Agilent's ChIP-on-chip System |
Comprehensive genome-wide and focused coverage
Each Agilent ChIP-on-chip microarray features a total of ~244,000 60-mer oligonucleotide probes. Probes are spaced every ~~100-300 bp across regions of interest in both coding and non-coding DNA sequence. Available formats include whole genome, selected, and focused promoter regions. Such focused microarrays include Proximal Promoter (-0.8 KB upstream to +0.2 KB downstream of identified transcriptional start sites) and Expanded Promoter (-8.0 KB upstream to +2.0 KB downstream) designs.
Superior microarray performance and capability
Proprietary microarray technology using optimized 60-mer oligonucleotide probes and a convenient two-color labeling system delivers higher sensitivity, accuracy, and greater reproducibility than one-color systems. These unique features allow sensitive measurements of weak- and infrequent-binding events, as well as direct comparisons of samples on the same microarray.
Greater confidence in binding event data
Powerful algorithms employ neighborhood probe voting with multiple probes to generate reliable data with greater true-binding events and fewer false positives.
Flexible and versatile user-defined microarray formats
The inherent flexibility of our microarrays allows users to define content - up to 244K customizable features per microarray - for any tiling density, genomic region, and organism of interest.
Quick turnaround
Agilent's SurePrint technology features a flexible, industrial-scale inkjet printing process that synthesizes oligonucleotide probes in situ onto 1" x 3" slides. The maskless process allows quick iteration of microarray designs in response to fast-paced content changes in the continuously evolving genomics environment. This allows researchers easy access to high-quality arrays loaded with relevant and rich content.
Easy-to-use data analysis software
ChIP Analytics Software combines annotated, algorithmic array data processing with an easily manipulated text file output and high-speed statistical modeling functions.
Access to probe sequence and annotation information
Agilent gives you complete access to probe sequences and annotation files to facilitate searching of public databases and extraction of biological information.
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| Describing the ChIP-on-chip Workflow |
The ChIP-on-chip workflow includes sample prepartion of the protein-DNA complex, followed by hybridizing of the sample to the microarray for subsequent analysis.
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The ChIP-on-chip Workflow consists of multiple steps. It begins with preparation of protein-DNA complexes from starting samples, followed by hybridization of the resulting labeled pool of DNA fragments to a ChIP-on-chip microarray for subsequent analysis. Major steps include:
- Rapid fixation of cells chemically cross-links DNA binding proteins to their genomic targets in vivo.
- Cell lysis releases the DNA-protein complexes, and sonication fragments the DNA.
- Immunoprecipitation (IP) purifies the protein-DNA fragments, with specificity dictated by antibody choice.
- Hydrolysis reverses the cross-links within the released DNA fragments, and amplification is performed.
- Labeling pool of protein-DNA fragments.
- Hybridization of DNA onto Agilent-validated or user-customized microarrays featuring 60-mer oligonucleotide probes.
- Data analysis with a high-resolution array scanning to detect significant binding events and results comparison with gene annotation databases using Agilent ChIP Analytics software (see following section).
The ChIP-on-chip process enables the observation of a few to 50-fold the representation of protein-bound (occupied) DNA promoter fragments of interest in the final DNA pool. |
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| Agilent ChIP Analytics Software |
ChIP Analytics Software provides an intuitive user interface for visually exploring and analyzing data from Agilent ChIP-on-chip microarrays. It accepts data outputs from Agilent Feature Extraction software or Molecular Devices GenePix software and analyzes the significance of protein binding events without extensive processing and manipulation. The software takes advantage of the high-speed statistical analytic functions that include user-configurable heuristics for binding event identification based on p-values and adjacent probes, as well as inter- and intra-array intensity normalization, and error modeling.
ChIP Analytics features at a glance
- Simultaneous analysis of sets of microarrays with annotations
- Output reports containing probe, sequence, and gene detail
- Option of UCSC track report in .BED format for convenient viewing within the UCSC Genome Browser (human reference sequence based on NCBI Build 35, May 2004)
- QC report for convenient, at-a-glance visualization of key experimental elements
- Support for replicates
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Chip Analytics probe report |
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Binding data from ChIP Analytics viewed in UCSC Genome Browser |
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| Agilent ChIP-on-chip Microarrays |
To provide a valued solution in this emerging technology, Agilent Technologies has available an expanding set of ChIP-on-chip products. Available are Catalog ChiP-on-chip microarrays that include yeast whole genome and focused promoter regions of human (and mouse, coming soon) and Custom microarrays with Array Design IDs using probes Agilent probes, or probes of your choice, each printed using Agilent's SurePrint technology.
Agilent ChIP-on-chip Product Platform
Agilent DNA Methylation Product Platform