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
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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 Student’s 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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