Roche NimbleGen | DNA Methylation

DNA Methylation

Roche NimbleGen provides the most sensitive and specific DNA Methylation microarrays and services on the market. Methylated DNA regions are accurately mapped using a combination of affinity-based enrichment, such as Methylated DNA Immunoprecipitation (MeDIP) or the Methylated CpG Island Recovery Assay (MIRA), followed by microarray analysis. Roche NimbleGen offers several types of DNA Methylation arrays for a variety of organisms to meet your specific research needs:

Whole-Genome Designs cover all unique genomic regions in single arrays or multi-array sets and offer unbiased, comprehensive genome coverage for the most complete data sets possible.
Promoter Designs cover important regulatory regions. Deluxe Promoter arrays offer extensive, single-slide coverage of those regions including gene and miRNA promoters, and CpG islands, while promoter arrays cover only gene promoters.
Targeted Designs focus on CpG islands, RefSeq genes, ENCODE regions (up to 30 Mb), promoters of non-coding RNAs or the HOX clusters.
Custom Designs allow researchers to specify their regions of interest for a fine-tiling approach or design their own targeted probes for a tailored array solution.

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Advantages

Comprehensive Set of Array Designs Cover All Your Needs

Roche NimbleGen offers whole genome, promoter, and custom array designs, allowing you to choose the design that meets your research needs. Our whole-genome DNA methylation designs interrogate the non-repetitive regions of human, mouse, rat, and plant genomes at intervals ranging from <100bp to 255bp for unbiased discovery of methylated DNA regions. Arrays focused on biologically significant genomic regions are also available including promoter/CpG island array designs and promoter only designs aimed at well characterized promoter regions. For custom designs, researchers can specify their regions of interest for fine-tiling of genomic regions or promoters or design a tailored array with their own probe designs.

High-Resolution, High-Throughput Analysis of DNA Methylation

NimbleGen high density DNA methylation microarrays are available in three formats: 2.1M (2.1 million probes on a single glass slide), 3x720K (3 identical arrays per glass slide with 720,000 probes per array), 385K (385,000 probes on a single glass slide) and 4x72K (4 identical arrays per glass slide with 72,000 probes per array). The 2.1M high density microarrays enable coverage of the entire non-repetitive human genome at 100bp spacing using only 10 slides - greatly simplifying whole genome analysis. Now with the 3x720K and 4x72K arrays, you can increase sample throughput and lower the cost per sample for targeted DNA methylation analysis.

Figure 1

Figure 1: Detection of Differential DNA Methylation using the Three New Human DNA Methylation 2.1M Designs - MeDIP samples from OT (maternally-derived ovarian teratoma cells) and CHM (paternally-derived hydatidiform mole) cells were labeled with Cy5 and the control (input) DNAs with Cy3, pooled and technical replicates were hybridized to the three different human array designs. Raw data were analyzed using NimbleScan software and the P-values estimating the significance of enrichment were visualized using SignalMap software. The black horizontal bar indicate the regions tiled on the different array designs. The red box highlights the differentially methylated region between the OT and CHM samples, which was easily identified using all three array designs. The blue box highlights a region of DNA methylation detected on the whole-genome tiling design with 100bp probe spacing but not in the whole-genome economy design with 205bp probe spacing illustrating the greater detection sensitivity possible using arrays with 100bp probe spacing.

High Sensitivity and Specificity Provide Unparalleled Results

Roche NimbleGen's proprietary, light-mediated synthesis process produces high-density microarrays of long oligonucleotide probes (50-75mer). These long oligo arrays, when used in combination with high-stringency hybridization protocols, produce results of unparalleled sensitivity and specificity. In addition, because Roche NimbleGen performs DNA methylation array experiments using a two-color protocol, where input control and MeDIP samples are co-hybridized to the same array, inter-array variation is eliminated. As a result, NimbleGen DNA Methylation Arrays can readily detect as low as two methylated CpGs in a 500 bp fragment.

Easy-to-use Graphical View of Data Facilitates Discovery

Roche NimbleGen’s SignalMap software enables you to visually interpret your data and perform peak finding. SignalMap’s graphic representation of your data aids in the discovery of methylated or unmethylated DNA regions and well as differentially methylated DNA regions between samples. A free, 30-day demo version of SignalMap software is available for download.

Figure 2

Figure 2: Identification of DNA Methylation Using NimbleGen Arrays - From the raw data, NimbleGen generates scaled log₂-ratio data for IP/input. A statistical method is used to generate p-value enrichment data for each probe; peaks (methylated regions) are then generated based on the p-value data. NimbleGen also provides gene annotations and CpG island annotations.

Most Up-to-date Genome Builds Ensure the Most Accurate Results

NimbleGen DNA Methylation designs are based on the latest genome assemblies and sequence annotations to ensure comprehensive and accurate representation of the genome. In addition, you can continue to access array designs based on past genome data builds, which can be particularly useful for comparisons to prior studies.

Applications & References

Applications	References
DNA Methylation Defines Repression of Pluripotency in Progenitor Cells
Using a murine system that progresses from stem cells to lineage-committed progenitors to terminally differentiated neurons, DNA methylation and Polycomb-mediated histone H3 methylation (H3K27me3) were analyzed. Several hundred promoters, including pluripotency and germline-specific genes, become DNA methylated in lineage-committed progenitor cells, suggesting that DNA methylation may already repress pluripotency in progenitor cells. Loss and acquisition of H3K27me3 was detected at additional targets in both progenitor and terminal states. Many neuron-specific genes that become activated upon terminal differentiation are Polycomb targets only in progenitor cells. Promoters marked by H3K27me3 in stem cells frequently become DNA methylated during differentiation. These data suggest a model how de novoDNA methylation and dynamic switches in Polycomb targets restrict pluripotency and define the developmental potential of progenitor cells.	Mohn F, et al. “Lineage-Specific Polycomb Targets and De Novo DNA Methylation Define Restriction and Potential of Neuronal Progenitors,” Mol Cell. 2008 Jun 20;30(6):755-66. [Epub 2008 May 29].
Promoter DNA Methylation in the Human Genome
DNA methylation, RNA polymerase occupancy, and histone modifications were measured at 16,000 promoters in primary human somatic and germline cells. CpG-poor promoters are found to be hypermethylated in somatic cells, which do not preclude their activity. This methylation is present in male gametes and resulted in evolutionary loss of CpG dinucleotides. In contrast, strong CpG-island promoters are mostly unmethylated, even when inactive. Weak CpG-island promoters are distinct, as they are preferential targets for de novomethylation in somatic cells. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, it was observed that inactive unmethylated CpG-island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation.	Weber M, et al. “Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome,” Nature Genetics2007; 39(4):457-466.
Interdependence between Methylation and Transcription in Arabidopsis thaliana
DNA methylation was mapped in the entire Arabidopsis thalianagenome at high resolution. DNA methylation covers transposons and is present within a large fraction of A. thalianagenes. Methylation within genes is conspicuously biased away from gene ends, suggesting a dependence on RNA polymerase transit. Genic methylation is strongly influenced by transcription: moderately transcribed genes are most likely to be methylated, whereas genes at either extreme are least likely. In turn, transcription is influenced by methylation: short methylated genes are poorly expressed, and loss of methylation in the body of a gene leads to enhanced transcription. These results indicate that genic transcription and DNA methylation are closely interwoven processes.	Zilberman D, et al. “Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription,” Nature Genetics2007; 39(1):61-69.
Genome-wide DNA Methylation of CpG Islands
Tiling arrays were used in combination with the methylated CpG island recovery assay (MIRA) to investigate methylation of CpG islands genome-wide. All four HOX gene clusters were found on chromosomes 2, 7, 12, and 17, which are preferential targets for DNA methylation in cancer cell lines and in early-stage lung cancer. Altogether, more than half of all CpG island-associated homeobox genes in the lung cancer cell line A549 were methylated. Comparison with ENCODE-derived data shows that the lack of methylation at CpG- rich sequences correlates with presence of the active chromatin mark histone H3 lysine-4 methylation in the HOXA region. Methylation analysis of HOXA genes in primary squamous cell carcinomas of the lung led to the identification of the HOXA7- and HOXA9-associated CpG islands as frequent methylation targets in stage 1 tumors. Widespread methylation of homeobox genes lends support to the hypothesis that a substantial fraction of genes methylated in human cancer are targets of the Polycomb complex.	Rauch T, et al. “Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay,” Proc. Natl. Acad. Sci. USA2007; 104(13):5527- 5532.
Large-scale Mapping of MeCP2-binding Sites
Mutations in MECP2 cause the autism-spectrum disorder Rett syndrome. MeCP2 is predicted to bind to methylated promoters and silence transcription. However, the first large-scale mapping of neuronal MeCP2-binding sites on 26.3 Mb of imprinted and nonimprinted loci revealed that 59% of MeCP2-binding sites are outside of genes and that only 6% are in CpG islands. Integrated genome-wide promoter analysis of MeCP2 binding, CpG methylation, and gene expression revealed that 63% of MeCP2-bound promoters are actively expressed and that only 6% are highly methylated. These results indicate that the primary function of MeCP2 is not the silencing of methylated promoters.	Yasui DH, et al. “Integrated epigenomic analyses of neuronal MeCP2 reveal a role for long-range interaction with active genes,” PNAS, 2007; 104:19416-19421

Workflows

DNA Methylation Delivery Workflow

Purchase a NimbleGen catalog array or custom designed array of your choice.
Prepare your MeDIP samples in your laboratory.
Label the immunoprecipitated (IP) and Input DNA samples with Cy5 and Cy3 respectively, using the NimbleGen Dual-Color DNA Labeling Kit.
Pool and hybridize the samples to the array using a NimbleGen Hybridization Kit and NimbleGen Hybridization System 4 or 12.
Wash the arrays using the NimbleGen Wash Buffer Kit and the NimbleGen Microarray Dryer.
Scan the array using the 2-μm, high-resolution NimbleGen MS 200 Microarray Scanner.
Analyze the data using NimbleScan software and visualize your MeDIP-chip results using SignalMap software.

DNA Methylation Service Workflow

Purchase NimbleGen catalog arrays or custom designed arrays of your choice.
Prepare your MeDIP samples in your laboratory and then ship them to Roche NimbleGen.
Roche NimbleGen will manufacture and process the arrays from labeling through data analysis.
Roche NimbleGen will send you raw and analzyed data, genome annotation, and promoter reports.
Visualize your results using SignalMap software.

Availability

Delivery and Service Workflows are not available for every DNA Methylation array design. Please consult the Availability section on this page for a complete list.

Availability

The 2.1M, 3x720K, 385K, and 4x72K array formats and the Delivery and Service workflows are not available for every DNA Methylation array design. Use the table below to identify the availability of different formats and workflows across our DNA Methylation design offerings.

Whole-Genome Designs

	2.1M	3x720K	385K	4x72K
A. thaliana	N/A	N/A		N/A
Human		N/A	N/A	N/A
Mouse		N/A	N/A	N/A
Rat		N/A	N/A	N/A
Custom

Promoter Designs

	2.1M	3x720K	385K	4x72K
A. thaliana	N/A	N/A		N/A
Human				N/A
Mouse				N/A
Rat	N/A			N/A
Custom

Targeted Designs

	2.1M	3x720K	385K	4x72K
Dog	N/A	N/A		N/A
Human	N/A	N/A
Mouse	N/A	N/A		N/A
Rat	N/A	N/A		N/A
Custom

KEY:
	- Delivery OR Service Workflow
	- Delivery Workflow Only
	- Service Workflow Only
N/A	- Not Available

Literature

Brochures & Datasheets

NEW! NimbleGen ChIP-chip & DNA Methylation Microarrays
Brochure (PDF Format 6MB)
NimbleGen DNA Methylation 2.1M Whole-genome Tiling and Deluxe Promoter Arrays
Datasheet (PDF Format 1.4MB)
NimbleGen DNA Methylation 4x72K Array Delivery
Datasheet (PDF Format 312KB)

User Guides

NimbleGen Arrays User’s Guide: DNA Methylation Analysis (Version 7.2)*
User’s Guide (PDF Format 1.5MB)
* DO NOT reference this User's Guide if you are performing sample labeling using the discontinued version of the Dual-Color DNA Labeling Kit (Catalog Number: 05223547001) . The labeling protocols in this User Guide WILL NOT WORK with the discontinued kit. To access the labeling protocols for discontinued DNA Labeling Kits go to the DNA Labeling Kits Product Notice page and download the previous version of the User Guide.
Guide to Functionally Annotating Your DNA Methylation Data (Version 1.2)
DAVID User’s Guide (PDF Format 274KB)
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Downloads

385K HG18 Tiled Promoters
List of HG18 genes whose promoters are tiled on 385K Promoter Arrays (Excel Format 13.2MB)
385K MM8 Tiled Promoters
List of MM8 genes whose promoters are tiled on 385K Promoter Arrays (Excel Format 11.3MB)

Application Notes & Whitepapers

NEW! NimbleGen arrays and LightCycler® 480 System: A complete workflow for DNA methylation biomarker discovery and validation
Application Note (PDF Format 1.7MB)
Frequent switching of Polycomb repressive marks and DNA hypermethylation in the PC3 prostate cancer cell line
Application Note (PDF Format 914KB)
Genome-Wide Screen of Promoter Methylation Identifies Novel Markers in Melanoma
Application Note (PDF Format 2.7MB)

For a complete listing of literature covering all Roche NimbleGen products and services please visit our literature page.

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