Balkan Journal of Medical Genetics

ALTERATIONS OF COPY NUMBER OF METHYLATION PATTERN IN MISMATCH REPAIR GENES BY METHYLATION SPECIFIC-MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION IN CASES OF COLON CANCER
Onrat ST1*, Çeken I2, Ellidokuz E3, Kupelioğlu A4
*Corresponding Author: Serap Tutgun Onrat, Department of Medical Genetics, Afyon Kocatepe University Medical Faculty, ANS Arastırma Uygulama Hastanesi, Morfoloji Binası, Ozdilek yolu, Afyonkarahisar, 03200, Turkey; Tel.: +90-272-246-3301, Fax: +90-272-246-3300, E-mail: tutgunonrat@ yahoo.com, sonrat@aku.edu.tr
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MATERIALS AND METHODS

Patients and Clinical Data. For this study, samples were retrieved from the Güneş Pathology Laboratory at Alsancak, Izmir, Turkey. Colorectal cancer tissue samples embedded in paraffin were used in our study. Seventy patients comprised our study population, 41 males (58.6%) and 29 females (41.4%). The mean age of the males was 55.02 ± 2.39 and 61.76 ± 12.58 for the females, (p = 0.029, <0.05). According to the age groups, 60<38 (54.3%) and 60>32 (45.7%) patients suffered with colorectal cancer. Of the 70 colorectal cancer specimens tested, the mean age of patients with adenocarsinomas was 61.63 ± 11.45 and those with carcinomas was 48.91 ± 11.57. Adenocarcinoma was found in 49 (70.0%) and adenoma in 21 (30.0%) of the total 70 colorectal cancer speciments. Paraffin-Embedded DNA Extraction. Slides with a slice of paraffin-embedded tissue (5 ×·5 mm, 10 mm thick) were heated for 30 min. at 70°C to melt the paraffin and then placed in fresh Xylol for 5 min. until the paraffin oil was completely dissolved. The slides were then kept for 30 seconds each in 99.96 and 75% ethanol and tap water, and finally placed in 1 M NaSCN at 37°C overnight. They were then washed with TE buffer [10 mM Tris-HCl (pH 8.5) and 1 mM EDTA] and air dried; 20-40 μL of Proteinase K solution [2 mg/ml recombinant Proteinase K (Roche Diagnostic, Mannheim, Germany) in 25 mM Tris-HCl (pH 8.2)] were then applied to the tissue, which was then transferred to a 1.5 mL tube containing 100 mL Proteinase K solution and incubated overnight at 37°C. After in incubation at 80°C for 20 min., the tubes were centrifuged for 10 min. at 13,000 rpm on an Eppendorf microcentrifuge. Finally, 2 mL of the supernatant was used for each methylation specific-multiplex ligationdependent probe amplification (MS-MLPA) reaction.

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