ely 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, 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 protocol. We used the Bioconductor lumi package, which was developed by our collaborator and is ” widely used as one of the standard tools to process both Illumina DNA methylation and mRNA expression data. The data first went through a QA/QC step. For Illumina expression data, the data passing QA step was preprocessed using a variance stabilization transformation method followed by quantile normalization. For methylation data, we first performed a color balance adjustment of methylated and unmethylated probe intensities between two color channels using a smooth quantile normalization method. The methylated and unmethylated probe intensities were then normalized using the SSN method. The methylation M-value was calculated to estimate the methylation level of the measured CpG sites. The follow-up analysis was then based on the M-value. We used a shift and scaling normalization method, which includes global background shift during normalization instead of more aggresive quantile normalization as described in reference 45. We made this decision primarily because we produced high quality and consistent data evident by the principal component analysis that we are now incorporating in the supplemental section. After preprocessing, the differential analysis of methylation data was similar to that used for expression microarray data. Probes or CpG-sites with all samples “Absent” were removed from 10 Genome-Wide DNA Methylation in Uterine Leiomyoma further analysis to reduce false positives. To compare the differences in both methylation and expression between leiomyoma and myometrial tissues, we performed differential analyses using routines implemented in the limma package. To ensure both high statistical BAY41-2272 price significance and strong biological effects, we require that the differentially methylated CpG sites had an FDR,0.01 and fold-change of.2; using this process 1031 CpG sites were identified. For mRNA expression data, we required that the differentially expressed genes had an FDR,0.01 and a fold-change of.1.5; with these parameters, we identified 525 genes. We mapped the d