Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform

DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation. However, accurate and reproducible quantification of DNA methylation remains challenging. In this report, we describe Bio-COBRA (combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform), as a novel approach to quantitative DNA methylation analysis. The combination of a well-established method, COBRA, which interrogates DNA methylation via the restriction enzyme analysis of PCR-amplified bisulfite treated DNAs, with the Bioanalyzer platform allows for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. The sensitivity and reproducibility of Bio-COBRA make it a valuable tool for the analysis of DNA methylation in clinical samples, which could aid in the development of diagnostic and prognostic parameters with respect to disease detection and management

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells

Assessment of DNA methylation in clinical CLL samples and a human lung cancer cell line treated with 5-aza-2′dC

<p><b>Copyright information:</b></p><p>Taken from "Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform"</p><p>Nucleic Acids Research 2006;34(3):e17-e17.</p><p>Published online 7 Feb 2006</p><p>PMCID:PMC1361623.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Methylation levels of TWIST2 in 19 primary CLL samples generated by Bio-COBRA and Southern blot. The correlation coefficient between the two data sets was 0.98. ( and ) Restriction digestions of SALL3 (B) and C/EBPα (E) in A549 cells treated with 5-aza-dC at six different concentrations for 72 h (concentrations are indicated at the top). ( and ) Bio-COBRA quantification of the restriction digestions shown in (B) and (E). As expected, low doses of the demethylating agent showed a pronounced effect in the DNA methylation status of the analyzed loci. ( and ) mRNA expression level of SALL3 (D) and C/EBPα (G). Three separate measurements were performed for each sample. For C/EBPα, the expression level measured in the untreated cell line was normalized to 1. For SALL3, the expression level detected at 0.10 µM was normalized to 1, since the untreated cell line shows no expression under the experimental conditions utilized in this study

Plots of observed versus expected DNA methylation values for SALL3, TWIST2 and C/EBPα methylation standards

<p><b>Copyright information:</b></p><p>Taken from "Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform"</p><p>Nucleic Acids Research 2006;34(3):e17-e17.</p><p>Published online 7 Feb 2006</p><p>PMCID:PMC1361623.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () SALL3, () TWIST2 and () C/EBPα results. Trend lines and values are displayed for each plot. The non-linearity of the observed versus expected methylation values is most likely due to a PCR amplification bias