Patient samples and controls

A total of 98 FFPE tissue samples from breast cancer patients, related to years 2005 to 2009, were collected from the Department of Pathology at the Imam Khomeini and Shafa University Hospitals, in Ahvaz, Iran. The samples were identified as ductal carcinoma in situ (DCIS) (n = 8), invasive ductal carcinoma (IDC) (n = 65), invasive and in situ ductal carcinoma (DCIS + IDC) (n = 20), invasive lobular and ductal carcinoma (ILC + IDC) (n = 2), and inflammatory carcinoma (n = 3). In addition, 10 fresh frozen normal breast tissue samples from the marginal tissues of tumor in breast cancer patients were obtained and analyzed. Ethical guidelines were met for sample collection.

EpiTect Methylated and Bisulfite converted Control DNA (Qiagen, Germany) and EpiTect Unmethylated and Bisulfite converted Control DNA (Qiagen, Germany) were used as positive (100% methylated) and negative (0% methylated) controls, respectively. Also EpiTect Unmethylated and Unconverted Control DNA (Qiagen, Germany) were used as the control for bisulfite modification.

A series of standard dilutions of methylated DNA was prepared by diluting 100% methylated and bisulfate-treated control DNA on a background of unmethylated and bisulfate-treated control DNA in ratios of 75, 50, 25, and 10%.