Thirteen normal colon epithelial tissues (NN) were obtained from patients without selleck chem inhibitor cancer from the Department of Pathology, The Johns Hopkins University. Stool gDNA from colon cancer patients, patients without cancer, and healthy control subjects were kindly provided by OncoMethylome Sciences (Sart-Tilman, Belgium). Written informed consent was obtained from the patients who provided the colon epithelial tissues and the stool gDNA, and this study was approved by the Institutional Review Board of the Johns Hopkins University in US and Vrije Universiteit Medisch Centrum in Belgium. DNA purification from stool DNA and bisulfite treatment Stool specimens were collected and immediately submerged in stool stabilization buffer (Exact Sciences, MA) and stored at room temperature until processing (within 72 hrs).

For recovery of human DNA, whole samples were homogenized in excess volume (17) of stool homogenization buffer (Exact Sciences, MA) and aliquoted in portions of 32 ml (the equivalent of 4 g of stool). Each aliquot of stool samples were centrifuged and the supernatants were incubated with RNase A for 1 hr at 37��C. The DNA was then precipitated with sodium acetate (pH 5.2) – isopropanol and washed with 70% ethanol. The DNA pellet was subsequently re-suspended in 4 ml of 1�� TE (pH 7.4) and proteinase K digested by the use of 400 ��l of 10X buffer (240 mM EDTA, 750 mM NaCl, pH 8.0), 400 ��l of 10% SDS and 20 ��l of proteinase K (20 mg/ml), and samples were incubated overnight at 48��C with constant shaking.

After centrifugation, 5 ml of phenol/chloroform/isoamylalcohol was added and samples were incubated (with shaking at 225 rpm) for 10 min at RT. After centrifugation again, the aqueous layer was transferred into new tubes, and DNA was precipitated and washed. Pellets were re-suspended in 2 ml of 1�� TE solution (pH 8.0). Bisulfite treatment of 1 ��g of tissue gDNA was performed to convert unmethylated cytosines to uracils for methylation analysis. For stool DNA, an up-scaled DNA modification step was applied to 32 ��g of the obtained DNA, using the EZ-96DNA Methylation Kit (Zymo Research, Los Angeles, CA), according to the manufacturer’s protocol. Bisulfite-treated DNA was concentrated using the Clean and Concentrator Kit (Zymo Research) and eluted in 30 ��l.

Sequencing and Combined Bisulfite Restriction Analysis (COBRA) All PCR reactions were done as described previously [14], and the primer sequences of bisulfite-DNA amplification were described previously [12]. PCR products were gel-extracted (Qiagen, Valencia, CA) and sequenced with an internal primer (F2) or forward primer (F1) using the ABI BigDye cycle sequencing kit (Applied AV-951 Biosystems, Foster City, CA). Searches for CpG islands in each gene promoter were done by using the online accessible software Methprimer. Bisulfite-sequencing primers were designed at the CpG islands within 1 or 2 kb upstream of the transcription start site (TSS).