Abstract
Over the last decade, the rapidly expanding interest in the involvement of DNA methylation in developmental mechanisms, human diseases, and malignancies has highlighted the need for an accurate, quantitative, and high-throughput assay. Existing methods are limited and are often too laborious for high-throughput analysis or inadequate for quantitative analysis of methylation. Recently, a MassCLEAVE assay has been developed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to analyze base-specific methylation patterns after bisulfite conversion. To find an efficient and more cost-effective high-throughput method for analyzing the methylation profile in breast cancer, we developed a method that allows for the simultaneous detection of multiple target CpG residues by using thymidine-specific cleavage mass array on matrix-assisted laser desorption/ionization time-of-flight silicon chips. We used this novel quantitative approach for the analysis of DNA methylation patterns of four tumor suppressor genes in 96 breast tissue samples from 48 patients with breast cancer. Each individual contributed a breast cancer specimen and corresponding adjacent normal tissue. We evaluated the accuracy of the approach and implemented critical improvements in experimental design.
Highlights
DNA methylation is an important potential biomarker in cancer study and opens a new area in cancer therapy. DNA methylation analysis is a rapidly developing field, studies about its clinical usefulness are limited due to the fact that no single technique is superior [1,2,3,4]
In the C-cleavage reaction, methylated regions are cleaved at every C to create fragments containing at least one CpG site each
On the MALDI-TOF MS, mass spectrum information can be used to determine methylated and unmethylated DNAs to assess the degree of methylation for each CpG island independently, and to estimate the average methylation for the entire target region [5, 6, 13, 14]
Summary
DNA methylation is an important potential biomarker in cancer study and opens a new area in cancer therapy. DNA methylation analysis is a rapidly developing field, studies about its clinical usefulness are limited due to the fact that no single technique is superior [1,2,3,4]. DNA methylation is an important potential biomarker in cancer study and opens a new area in cancer therapy. A novel EpiTYPER assay for high-throughput analysis of DNA methylation patterns using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently been introduced [5, 6]. This assay is a tool for the detection and quantitative analysis of DNA methylation using MALDI-TOF MS and MassCLEAVE reagent, which enables base-specific (C/T) cleavage reactions [5, 7, 8]. In a completely new and comprehensive study, by quantitative analysis of methylation patterns in a set of >400 candidate genes in 59 different cancer cell lines, the developer company showed robustness of (C/T) cleavage reactions in methylation studies [6]
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