Genomics

Dataset Information

34

Proof of principle of novel technique of methylation analysis


ABSTRACT: In this work we demonstrate a novel method of methylation detection that utilises immunoaffinity to detect the presence of methylated DNA hybridised to a cDNA microarray. We use a monoclonal antibody specific to 5 methylcytosine to detect the presence of 5 methylcytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2900 genes show the presence of methylation in this condition. We also report that 165 genes are consistently methylated in all replicates of these experiments. The methylated genes show a uniform distribution over all the chromosomes. The gene ontology of these also indicates no functional correlation between the genes that are methylated. We detect the presence of methylation in IGF2, an imprinted gene and thus known to harbour DNA methylation. The method is extremely specific and offers a quick and efficient way to analyse the methylation landscape on a high throughput scale. This method uses existing technology to assess methylation and thus can integrate very efficiently into any platform used. A method that detects DNA methylation that is not restricted to specific sequences would provide a useful platform to analyse methylation distribution among the genes in any given phenotypic condition.Availability of additional approaches to examine DNA methylation would lead to increase in our understanding of the methylome and would help define the intrinsic and extrinsic factors which govern it. Microarray based high throughput methods have been extensively used for gene expression analysis. The method described uses a microarray to analyse DNA methylation levels in a gene/cDNA/oligo specific manner. Here, genomic DNA is fragmented and used for hybridization to microarray slides. The presence of DNA cytosine methylation in the hybridized DNA is detected by employing Cy3 labelled anti 5mC( 5-methyl cytidine) antibody (monoclonal). The resolution provided by this method is dependent upon the microarray platform used. This method can be adapted to any platform thus enabling seamless integration with existing technology of gene expression analysis Overall design: The experiment involves the use of genomic DNA from human fibroblasts with the karyotype 45XO and 47XXX. This experiment was performed to analyse the methylation distribution in the human genome in the absence of X chromosome inactivation and this was compared to the methylation distribution in the genome in the background of XCI. 3 technical replicates were used for the XO sample and 2 for XXX. The methylation was estimated by calculating the fold florescence intensity to control spots for the single channel experiments carried out. Each replicate was normalised individually with the background florescence. The normalised florescence values were used for further comparison between and within replicates.

INSTRUMENT(S): UHN Homo sapiens 19.2K cDNA array

SUBMITTER: Ashwin Kelkar  

PROVIDER: GSE20873 | GEO | 2010-04-09

SECONDARY ACCESSION(S): PRJNA124665

REPOSITORIES: GEO

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Publications

A novel method to assess the full genome methylation profile using monoclonal antibody combined with the high throughput based microarray approach.

Kelkar Ashwin A   Deobagkar Deepti D  

Epigenetics 20090818 6


In this work we demonstrate a novel method of methylation detection that utilises immunoaffinity to detect the presence of methylated DNA hybridised to a cDNA microarray. We use a monoclonal antibody specific to 5 methyl cytidine to detect the presence of 5 methyl cytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2,900 genes show the presence of methylation in this condition. We also report that 165 genes are consistently methylated in all replicates  ...[more]

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