APOPTOSIS GENE EXPRESSION PROFILE IN EARLY-STAGE NON SMALL CELL LUNG CANCER
Metodieva SN1, Cherneva RV, Nikolova DN, Genchev GD, Petrov DB, Toncheva DI
*Corresponding Author: Svetlana Nikolova Metodieva, Department of Medical Genetics, Medical University Sofia, 2 Zdrave str, 1431 Sofia, Bulgaria; Tel.: +359-2-952-0357; Fax: +359- 2-952-0357; E-mail: svetlana.metodieva@yahoo.com
page: 47

MATERIALS AND METHODS

The study includes 12 patients with primary NSCLC who were admitted to the Clinic of Thoracic Surgery, St. Sofia University Hospital, Sofia, Bulgaria, between November 2007 and July 2008 and underwent a lobectomy for resection of tumor nodules. None had received prior therapy. Tissue acquisition was approved by the Institutional Ethics Committee and all participants signed informed consent forms. We analyzed 12 tumor samples and eight adjacent non cancerous lung tissues (as controls). All tumors were staged postoperatively according to the classification system of the International Union Against Cancer (UICC) and were early-stage NSCLCs. Five patients had squamous cell carcinoma (SCC) and seven patients had adenocarcinoma (AC). Clinical characteristics of the patients are summarized in Table 1. Total RNA was extracted from the tissue samples (RNeasy MiniKit; Qiagen, Hilden, Germany) and genomic DNA contamination was eliminated (RNAse-free DNAse set; Qiagen). RNA concentration was measured spectrophotometrically and the RNA integrity of all samples was tested by denaturing agarose electrophoresis. One μg of each RNA sample was used for cDNA synthesis (High Capacity Reverse Transcription Kit; Applied Biosystems, Foster City, CA, USA). Polymerase chain reaction (PCR) was performed on a 7500 Real Time PCR System (Applied Biosystems). Gene expression was analyzed using Human Apoptotic RT2 Profiler PCR Array (SuperArray Bioscience Corporation, Qiagen, Hilden, Germany). For expression analyses we used webbased software (available at www.sabiosciences. com), which automatically performs fold-change calculations based on the ΔΔCt method. The house-keeping gene RPL13A was used for data normalization. For each tumor, the fold change of each gene was calculated compared to the mean value of its expression in the control group. A gene was considered to be down or up regulated if its expression was altered more than 4-fold in at least half of the analyzed NSCLCs. For statistical evaluation of the data we used the Studentís t-test and the non parametrical tests of Shapiro-Wilk and Mann-Whitney. A p-value of 0.05 was accepted as the threshold for significant difference between the tumor and non tumor samples (Table 2).



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