POLYMORPHISMS OF HPC2/ELAC2 AND SRD5A2 (5α-Reductase Type II) GENES IN PROSTATE CANCER
İzmirli M1,*, Arikan B2, Bayazit Y3, Alptekin D4
*Corresponding Author: Muzeyyen İzmirli, Department of Medical Biology, Faculty of Medicine, Bezmialem Vakif University, 34093, Istanbul, Turkey; Tel.: +90-212-523-37-19, Fax: +90-212-523-23-26; E-mail: muzeyyenizmirli@gmail.com.
page: 31

MATERIALS AND METHODS

We studied 64 prostate cancer patients (mean age 65.24 8.63) who had adenocarcinoma and 34 healthy controls (mean age 49.83 18.29) without a family history of cancer. All came from the Çukurova region of southern Turkey. Their clinical data were on record at Çukurova University, Faculty of Medicine Hospitals, Adana, Turkey. The research protocol was approved by the Ethical Committee of Çukurova University, Faculty of Medicine. The obtained venous blood samples were collected into CBC tubes and stored at 4C. DNA was extracted from whole blood samples using the salting-out procedure [8]. For the Ser217Leu polymorphism in the HPC2/ ELAC2 gene, the polymerase chain reaction (PCR) amplification used primers and conditions as described in [4]. The 276 bp PCR product was digested overnight with TaqIα at 65C. Subsequently, genotypes were visualized on a 10% polyacrylamide gel. For the Ala541Thr polymorphism of the HPC2/ ELAC2 gene, the PCR amplification used primers and conditions as previously described in [4]. The 419 bp PCR product was digested for 3 hours with Fnu4HI at 37C. Genotypes were visualized on a 10% polyacrylamide gel. For the Ala49Thr and Val89Leu polymorphisms in the SRD5A2 gene, the PC reactions were carried out using a master mix that consisted of 2.5 μL of 10X PCR buffer, 0.2 μL of 25 mM dNTPs, 0.2 μL of each 20mM primer, and 1 μL Taq polymerase (5U/ μL) and ddH2O, for a total reaction volume of 25 μL. Nested PC reactions were used to amplify the regions containing the polymorphisms of interest. The forward (5-TGG CCT TGT ACG TCG CGA AG-3) and reverse (5-AGC AGG GCA GTG CGC TGC ACT-3) primers were used to amplify the region containing the Ala49Thr and Val89Leu polymorphisms. These were amplified with a 35-cycle protocol at 95C for 3 min. for one cycle; 96C for 30 seconds, 62C for 1 min., and 72C for 1 min. for 35 cycles; followed by an elongation cycle at 72C for 10 min. Both primers amplified with a 261 bp region. Finally, 5 μL of the final 276 bp PCR product was digested with MwoI and RsaI overnight at 65C. Genotypes were visualized on a 10% polyacrylamide gel and stained in ethidium bromide for 5 min., visualized using the UviTech Gel documentation system and then evaluated [9]. Statistical analyses of the data were performed by a SPSS (11.5 version) program. The Pearson Chi- Square test was used to compare the ratios, and values of p <0.05 were considered statistically significant.



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