e89 Nucleic Acids Research, 2009, Vol. 37, No. 12 PAGE 4 OF 11 Figure 1. Schematic of the procedure. Shown at the top is genomic DNA with a CpG island that is methylated. The DNA is cleaved with the restriction endonuclease MspI and adaptors ligated. The ligated material is divided evenly, one half being digested with McrBC and the other half being mock digested. This material is used as template for PCR ampliﬁcation and the resulting product is used for microarray comparison. 1 million of the 1.7 million CpG dinucleotides occurring in CpG islands, much more than can be queried by other techniques that utilize diﬀerent enzymatic depletions such as HpaII and MspI ( 225 000). This gives our meth- odology a 4-fold increase in potential coverage over other methods. According to these calculations combined with the increased array coverage (where we include non-pro- moter sequences and do not discriminate against any other promoter sequences except for larger islands, see ‘Methods for array design’ section) we will have increased the level of DNA methylation that can be measured in the genome over other methods which either use restriction enzymes or limited array coverage (data not shown). The procedure as schematized in Figure 1 involves digesting the genome with a restriction endonuclease with a CG-rich recognition sequence (MspI), and ligation of adaptors for use in a subsequent step of reducing geno- mic complexity. We next divide the ligation in half and deplete one-half of its methylated sequences by digestion with the methylation-speciﬁc endonuclease, McrBC (37), and mock treat the other half. In both cases, we use care- fully balanced PCR conditions to size select MspI frag- ments and reduce the overall genome complexity as previously described (29). The McrBC-treated representa- tion is compared to the mock-treated sample that serves as the reference for comparative hybridization to the designed oligonucleotide array. To determine if our method accurately identiﬁes the methylation state of CpG islands, we ﬁrst performed initial experiments with cell lines. To determine the level of noise in the system we performed analysis on a normal ﬁbroblast cell line (chp-skn-1). Two cultures of chp-skn-1 were grown and DNA prepared. Representations were prepared and were used to compare hybridization results from the same representations, representation from the same sample prepared separately, as well as the chp-skn- 1 grown separately. Generally two hybridizations are per- formed one being a dye swap to decrease variation but in this case four hybridizations were performed for all condi- tions; however to demonstrate how little noise there is in the system, results and correlation from a single hybridization were compared graphically shown in Figure 2A–C. The close correlation demonstrates the small variation in the representational process, the labeling and the hybridization. We also found little variation among diﬀerent lots of McrBC (data not shown). Included on the array are probes that are not in a repre- sentation but have moderate CpG density. Using the average intensity for these probes in the mock channel as the background component we measured signal to noise for the probes on the array. The average signal to noise for probes on the array was 10.39. We then performed a comparison of the breast cancer cell line SKBR3 with chp-skn-1, shown in Figure 2D (with a correlation of 0.831, which demonstrates that there are diﬀerences detected between two diﬀerent samples as com- pared to the control experiments shown in Figure 2A–C. In the scatter plot (Figure 2D), points oﬀ the central diag- onal represent fragments detecting diﬀerential methylation between the two samples. To evaluate the accuracy of measurements 25 fragments identiﬁed with methylation diﬀerences between chp-skn-1 and SKBR3, and nine frag- ments with no detection of methylation were selected for validation by sodium bisulﬁte sequencing (42). Analysis of one representative fragment is shown in Figure 2E. In each of the 28 fragments sequenced CpG dinucleotides inclusive ofo McrBC sites were mapped and their methy- lation state identiﬁed in the two samples (Supplementary Figure 1 for ﬁve non-gene islands, ﬁve non-CpG island regions and 10 gene-associated islands; Supplementary Table 1 which has data for all 28 fragments). Fragments were selected based on varying the diﬀerence in the array ratios measured for both samples. By doing so we were able to determine at what level accurate measurements of methylation can be determined. At a diﬀerence around 0.3 the measurements may be incorrect as can be seen by two fragments, which had a diﬀerence in ratio of 0.317. Any ratio diﬀerence above this level was found to be methy- lated in one of the samples. To determine if our method could accurately identify known tumor suppressor CpG island methylation, we then analyzed the hepatocellular cancer cell line HuH7 with known methylation of the p16 promoter (43). In Figure 2F we show the detection of methylation of the region of the p16 gene CpG island commonly methylated and correlated with decreased gene transcription. Finally, we compared the results of our methodology on the two cell lines HPDE and PANC1 with results from the same two cell lines reported by Sato and colleagues using a diﬀerent method and diﬀerent array design (44). Although there were some diﬀerences in detection between the two methods they were in good agreement (68%) for islands methylated in the samples. Of the 34 interesting islands listed and validated, 26 were represented on our array