marrow of breast cancer patients. Using genome-wide analyses of DNA methylation in uterine leiomyoma we hope to define a specific epigenetic profile that could inform the development of diagnostic biomarkers for uterine leiomyoma as well 9720791 as identify potential therapeutic targets. Because DNA methylation is reversible, epigenetic modifying drugs could be used in the medical management of uterine leiomyoma. Importantly, aberrant DNA methylation and other epigenetic abnormalities may represent a critical initial mechanism that triggers transformation of a single myometrial cell that will eventually give rise to a monoclonal leiomyoma tumor. Understanding the mechanism underlying the pathogenesis of uterine leiomyoma will be critical for developing new preventive and therapeutic approaches to the disease. Materials and Methods Ethics Statement To obtain human tissues, we followed the protocol approved by the Institutional Review Board for Human Research of Northwestern University and New York University. Written informed consent was received from all subjects. Tissue acquisition For in vivo studies, we obtained matched pairs of leiomyoma and adjacent myometrium from a total of 23 African American and 14 Caucasian-American subjects undergoing hysterectomy for symptomatic fibroids. To minimize heterogeneity due to race we used samples from 18 African American subjects for both genome-wide DNA methylation and gene expression microarrays. In follow-up verification studies, we included samples from 4 of the original African American group plus 4 additional Caucasian subjects for bisulfite sequencing and all 18 original African American plus 10 Caucasian subjects for mRNA quantification using real-time RTPCR. Samples from Caucasian subjects were added to evaluate whether similar patterns of ” DNA methylation and mRNA expression were observed. Key clinical characteristics of the 18 African American subjects, whose samples were used for both microarrays are described in Primary cell isolation Leiomyoma smooth muscle cells were isolated from the peripheral portions approximately 1 cm from the outer capsule of the leiomyoma, and then cultured as previously described with minor modifications. Cells were cultured in DMEM/F12 1:1 containing 10% fetal bovine Genome-Wide DNA Methylation in Uterine Leiomyoma serum and grown in a humidified atmosphere with 5% CO2 at 37uC. Primary cells were used only up to the second passage to avoid changes in phenotype and gene expression. DNA methylation and mRNA expression analysis Genomic DNA was isolated from 20 mg frozen tissues using the DNeasy Blood & Tissue. One microgram of genomic DNA from each sample was bisulfite-modified using 9 Genome-Wide DNA Methylation in Uterine Leiomyoma the EZ DNA Methylation kit, Danoprevir according to the manufacturer’s protocol along with the technical validation of the assay. Bisulfite-modified DNA was hybridized to the HumanMethylation27 Beadchip. Total RNA was isolated from 20 
mg of frozen tissues using the RNeasy Fibrous Tissue kit according to manufacturer protocols with minor modifications. After elution, RNA samples were quantified using a ND-1000 spectrophotometer and evaluated for degradation using a 2100 Bioanalyzer. For use in hybridization, samples were required to have a RIN.9, an OD260/280 of 1.92.0, and OD260/230.1.5, and a 28S:18S ribosomal band ratio of.1.5. The samples were hybridized into the HumanHT-12 v3 genome-wide gene expression BeadChips according to the manufacturer’s