Before nucleic acid extraction, the cryosections of frozen tissue specimens were stained with hematoxylin-eosin

and evaluated for tumor cell content. Only the tumor samples that contained at least 50% of tumor cells on a microscopic section were used for further processing. Consequently, 151 pairs of cancerous and matched unaffected lung tissues were selected for the study. Clinicopathologic data and previously detected EGFR, KRAS, and HER2 gene mutational status were available for all the patients. For survival analysis, the overall survival (OS) was estimated as the time from the date of the surgery to the date of death due to lung cancer recurrence or metastases (event) or to the date of the last control visit (censoring). The disease-free survival (DFS) was defined as the time from the date of the surgery to the date of disease ZD1839 nmr relapse or death, whichever occurred first (events), or to the date of the last visit (censoring). The study was approved by the Ethics Committee of the University, and written informed consent for specimen collection was obtained from each patient before the surgery. DNA and RNA were isolated simultaneously using a magnetic extraction method. Briefly, about 40 to 50 mg of tissue was disrupted in lysis buffer (Biomerieux, Marcy l’Etoile, France) with TissueRupter

(Qiagen, Hilden, Germany) and incubated with Proteinase K for 2 hours at 56°C. Nucleic acids from deproteinated cell lysates were extracted automatically on the EasyMag machine 17-AAG in vitro (bioMérieux) according to the producer’s protocol. Both DNA and RNA were present in the 100-μl resulting extracts. Nucleic acid quality was assessed electrophoretically. For gene expression analysis, RNA was transcripted into cDNA in a reaction with High Capacity RNA-to-cDNA

Master Mix (Applied Biosystems, Foster City, CA) according to the producer’s recommendations. MET CN was analyzed by a quantitative real-time duplex CYTH4 polymerase chain reaction (qPCR) on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) with a commercially available predesigned MET TaqMan Copy Number Assay (Hs0143282_cn) and a Reference RNase P Assay (PN4412907), both from Applied Biosystems. The qPCR was done in a 20-μl reaction mixture containing 10 μl of Applied Biosystems TaqMan Universal PCR Master Mix with UNG, 1 μl of the CN assay solution, 1 μl of the reference assay solution, and 5 μl of DNA solution according to the following cyclic conditions: 50°C for 2 minutes followed by holding for 10 minutes at 95°C and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Each sample was analyzed in quadruplicate. The raw post-PCR data were used for MET CN calculation by the relative quantification method using the CopyCaller v.1.