Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis

Abstract

Oligonucleotide microarray-based hybridization is an emerging technology for genome-wide detection of DNA variations. We have extended this principle and developed a novel approach, called methylation-specific oligonucleotide (MSO) microarray, for detecting changes of DNA methylation in cancer. The method uses bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. The amplified product, therefore, may contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. A test sample is hybridized to a set of oligonucleotide (19-23 nucleotides in length) arrays that discriminate methylated and unmethylated cytosine at specific nucleotide positions, and quantitative differences in hybridization are determined by fluorescence analysis. A unique control system is also implemented to test the accuracy and reproducibility of oligonucleotides designed for microarray hybridization. This MSO microarray was applied to map methylated CpG sites within the human estrogen receptor alpha (ERalpha) gene CpG island in breast cancer cell lines, normal fibroblasts, breast tumors, and normal controls. Methylation patterns of the breast cancer cell lines, determined by MSO microarray, were further validated by bisulfite nucleotide sequencing (P <0.001). This proof-of-principle study shows that MSO microarray is a promising technique for mapping methylation changes in multiple CpG island loci and for generating epigenetic profiles in cancer.

Figure 1

Schematic outline for analysis of DNA methylation based on oligonucleotide microarray. Genomic DNA is bisulfite treated and amplified by PCR for a specific CpG island region of interest. The amplified product is labeled with Cy5 fluorescence dye and hybridized to oligonucleotide probes attached to a glass surface. At left an oligonucleotide probe is designed to form a perfect match with a target DNA containing the unmethylated allele. At right a probe is designed to form a perfect match with the methylated DNA target.

Figure 2

(A) Nucleotide sequences of a downstream region of the human ERα CpG island (GenBank, accession no. X03635.1 G1:31233). The 15 CpG sites tested by MSO microarray are underlined and shown in bold. (B) Nucleotide sequences of methylated and unmethylated probes to be analyzed in MSO microarray.

Figure 3

Standardization curve for MSO assays. (A) Mixtures of in vitro-methylated and untreated control DNA were prepared and amplified by PCR using bisulfite primers for the human ERα CpG island (see Methods). Target DNA was hybridized to MSO microarray. The Cy5 red fluorescence signals of the methylated (M) and unmethylated (U) alleles for CpG#14,15 are shown, reflecting the indicated percentage of methylation. (B) A calibration curve for measuring methylation changes at the ERα CpG sites. The intensity ratios (Y-axis) represent signal intensities of log M/log M+log U. The linear distribution shows that measurements of the different mixtures are well distinguished and can be used to determine the methylation status for test samples. The MSO assays were independently repeated three times with different target preparations.

Figure 4

Methylation analysis of 15 CpG sites in the human ERα CpG island using MSO microarray. The nucleotide positions of these sites are shown in Fig. 2B. (A) The red fluorescence images are shown for MCF-7 and MDA-MB-231 targets. For MCF-7 images, no methylation has been detected in the interrogating sites. The same probe can differentially discriminate between M (methylated allele) and U (unmethylated allele) in the Cy5-labeled targets for MDA-MB-231 cells. Various degrees of methylation were detected in MDA-MB-231 cell lines. This experiment was independently performed three times with different target preparations. (B) Comparison of methylation levels deteteced by the methylation-specific oligonucleotide microarray with those derived from bisulfite sequencing. The histograms show the percentage of methylation levels of CpG sites analyzed in MDA-MB-231 cells. Note that the methylation levels measured by the MSO microarray showed a significant correlation with data obtained from bisulfite sequencing (P <0.001, assessed by linear regression analysis).

Figure 5

Methylation analysis of human ERα CpG island by MSO microarray. Summaries of the MSO results are shown for six breast cancer cell lines, one normal human fibroblast cell strain (HFF), four normal (N) breast tissue samples, and 15 primary breast tumors (T). Gray scale shown at right represents the methylation levels in percentage determined from the calibration curve for the test CpG sites (see example in Fig. 3).

Figure 6

MS–PCR analysis of the ERα CpG#7–11 in primary breast tumors (T) and normal breast tissue (N). M and U indicate amplification using methylated and unmethylated sequence-specific primers, respectively. (Pos) Positive control; (Neg) negative control.