Overview

Understanding the complex role and patterns of DNA methylation is a very active field of research around the world. DNA methylation is one of several modifications that normal DNA goes through after each replication. In vertebrates, DNA methylation typically occurs at CpG sites, where a cytosine is directly followed by a guanine in the DNA sequence. This methylation results in the conversion of the cytosine to 5-methylcytosine. CpG sites are often found at higher density near gene promoters where they are collectively referred to as CpG islands. The methylation state of these CpG sites can have a major impact on gene activity/expression. Neoplasia is often characterized by frequent abnormal increases or decreases in DNA methylation. If the genes affected by abnormal methylation happen to be involved in regulating cell proliferation, uncontrolled cell division can occur. In addition, the overall methylation state in a cell might also lead to chromosome instability and increased mutation rates, resulting in cancer.

Introduction

The Genotyping Division of the RPCI Microarray and Genomics Facility utilizes the Sequenom MassARRAY Compact system to offer high-performance methylation analysis. This MassARRAY EpiTyper platform utilizes mass spectrometry (MS) for the detection and quantitative analysis of DNA methylation using Homogeneous MassCLEAVE (hMC) base-specific cleavage and matrix-assisted laser desorption/ionization time-of-flight MS. The MassARRAY system is ideal for detection of methylation, for the discrimination between methylated and non-methylated samples, and for the identification of differentially methylated sites through quantitative analysis of methylation. Base-specific cleavage of 1ug bisulfite-treated DNA results in clear differences between the mass signal patterns of methylated and non-methylated DNA. Semi-quantitative methylation analysis is performed by comparing signals (the area under the peak) representing methylated and non-methylated DNA. Two base-specific cleavage reactions (T reverse and C reverse) are performed to discover methylation sites and to determine methylation ratios within a given target region. Depending on the sequence content, up to 85% of the CpGs within a target region are represented in the two cleavage reactions. The mass spectrometer is configured to accept two, miniaturized 384-pad SpectroCHIPs that can be sequentially analyzed in a single, automated run. Methylation calls are performed by the EpiTyper software v1.0 (Sequenom) and written to an Oracle 8i database to:

All applications are supported by SEQUENOM’s available hardware, software and proprietary SpectroCHIP consumables, which automatically generates analysis data and assigns methylation levels to CpG units. The Genotyping facility employs state of the art equipment including Qiagen, Matrix and Tecan robotic pipetting devices, 4 x 384 well PTC-225 thermalcycling machines, a Samsung chip spotting device, and the MassARRAY compact system for determining the absolute mass of short DNA sequences. This high-tech equipment forms the core of the genotyping facility and enables throughputs in excess of 10,000 reactions per day.

Our service includes primer and assay design through to the detection of methylation spectra.  All steps involved are highly automated and are tracked using a laboratory management system. In this method, CpG island PCR followed by IVT and RNase cleavage is performed in a single well. The size of the cleavage products are determined directly by the MassARRAY, yielding relative methylation measurements. Currently, the system permits the methylation status of up to 192 different CpG islands per sample in a single 384 well plate.

Project Design

Because of the unique characteristics of any methylation project and the high quality we aim for, we meet with each investigator who wishes to use our service. Primer and assay design will be carried out and a QC methylation run performed. A second meeting will occur after this QC run to discuss results. Depending on those results, we will either proceed with full-scale methylation analysis or consider alternative methods as necessary. Methylation results are provided to the user in an electronic format as an excel document (methylation ratios) and image (epigram).

Methylation failures

All customers must be aware that not all CpG islands/genes submitted can produce successful assays. At the PCR design stage, there is a drop-out rate of 10-15% of sequences which fail assay design completely. This can be due to the sequence surrounding the CpG site of interest containing repeats or CG rich regions. If it is possible to design an assay, there is then a further chance of the assay not generating good quality methylation data due to amplicon size, quality of bisulfite treated DNA and restrictive PCR conditions. In the past, we have seen up to a 10% further loss of methlaytion assays at this stage.

Our goal is to achieve >85% success rate in methlylation calls for a CpG site, with a minimum cut-off of >80%. When lower success rates are achieved, it is the user’s choice as to whether to have the data.

Methylation Analysis

Methlylation Analysis using Sequenom Mass Spectrometry is best suited for assaying 50s - 100s of samples for 10s-100s of CpG islands. Orders consisting of < 20 samples and < 5 CpG islands are more expensive per assay than larger projects because smaller sample sets do not fill all positions of the 384 pad, single use SpectroChip.   We require 1ug DNA (dried down in 96-W plates) and 1500-2000 bp of sequence (as indicated). Contact us for details.

The price for methylation analysis depends on the number of DNA samples and regions assayed.  A rough breakdown is described below:

Plate cost: ($1,100 per 384 samples) – As with all MassARRAY analyses, the SpectoChips hold 384 samples and are single use.  We recommend that you maximize the number of methylation reactions to reduce overall costs.  Each DNA samples requires two reactions (C & T-reverse), so 190 samples assayed for one CpG island will require one chip. 

Other combinations to fill one chip:  96 samples x 2 CpG regions, 48 samples x 4 CpG regions, 24 samples x 8 CpG regions, etc

Oligo and set up fee: ($50 per set) – Each CpG region assayed requires primers for PCR amplification.

Bisulfite treatment ($3 per individual sample, $175 per 96 samples) – The cost is @ $0.80 less per sample when utilizing the 96-W bisulfite kits.  1ug of bisulfite treated DNA will yield enough for 50 methylation reactions. You can certainly provide the BIS DNA if you would rather prepare it, but it must be freshly prepared and stored frozen.

These fees are internal RPCI investigator rates, outside users please inquire about non-RPCI pricing.

To use the Methylation service, we require that you electronically submit DNA samples (96-W plate format) and CpG island sequence (with identifier). After acceptance, we require a completed order form and 1ug DNA dried down in 96-W plates, with two wells remaining empty for inclusion of positive and negative controls.  DNA must be eluted in water as TE will interfere with the genotyping assay.