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A novel method to quantify local CpG methylation density by regional methylation elongation assay on microarray.

Zhang D, Wang Y, Bai Y, Ge Q, Qiao Y, Luo J, Jia C, Lu Z - BMC Genomics (2008)

Bottom Line: Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample.The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China. zdd_7597@seu.edu.cn

ABSTRACT

Background: DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.

Results: We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray), provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients.

Conclusion: This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

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MLH1, TERT and MGMT promoter methylation densities of tumor samples from 18 patients with colorectal tumor.
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Figure 4: MLH1, TERT and MGMT promoter methylation densities of tumor samples from 18 patients with colorectal tumor.

Mentions: In order to validate the accuracy of the RMEAM approach, we analyzed the MLH1, TERT and MGMT promoter methylation density in 18 colorectal carcinoma patients. Figure 4 showed that this assay could detect precisely the methylation density in every patient sample. In these samples, MLH1 promoter had 0%–5.21% methylation density (mean, 0.78%); TERT promoter had 1.18%–12.59% methylation density (mean, 4.48%) and MGMT promoter had 1.55%–30.84% methylation density (mean 14.44%). The region of MLH1 promoter in this assay was selected according to Maekawa et al [26]. This region is a little farther than the region relative to the transcriptional start site of MLH1 described by Deng et al. [27]. Consequently the methylation level of this region is relative low.


A novel method to quantify local CpG methylation density by regional methylation elongation assay on microarray.

Zhang D, Wang Y, Bai Y, Ge Q, Qiao Y, Luo J, Jia C, Lu Z - BMC Genomics (2008)

MLH1, TERT and MGMT promoter methylation densities of tumor samples from 18 patients with colorectal tumor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262069&req=5

Figure 4: MLH1, TERT and MGMT promoter methylation densities of tumor samples from 18 patients with colorectal tumor.
Mentions: In order to validate the accuracy of the RMEAM approach, we analyzed the MLH1, TERT and MGMT promoter methylation density in 18 colorectal carcinoma patients. Figure 4 showed that this assay could detect precisely the methylation density in every patient sample. In these samples, MLH1 promoter had 0%–5.21% methylation density (mean, 0.78%); TERT promoter had 1.18%–12.59% methylation density (mean, 4.48%) and MGMT promoter had 1.55%–30.84% methylation density (mean 14.44%). The region of MLH1 promoter in this assay was selected according to Maekawa et al [26]. This region is a little farther than the region relative to the transcriptional start site of MLH1 described by Deng et al. [27]. Consequently the methylation level of this region is relative low.

Bottom Line: Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample.The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China. zdd_7597@seu.edu.cn

ABSTRACT

Background: DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.

Results: We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray), provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients.

Conclusion: This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

Show MeSH
Related in: MedlinePlus