The inclusion criteria were as follows: (1) patients had a pathologically-confirmed diagnosis of NSCLC (2) and peripheral blood lymphocytes and FDG-PET images were available for analysis.
Patients had a LDN-193189 molecular weight standard staging work-up that included fibroscopy, a chest and abdominal CT scan, brain MRI or CT imaging, and FDG-PET. One hundred fifty-four patients with NSCLC met the inclusion criteria with a median follow-up time of 7.5 months (range, 0.13 – 29.5 months). There were 62 deaths (40.3%) during the study period. Torin 2 order Single nucleotide polymorphism Selection Single nucleotide polymorphisms (SNPs) were chosen for non-synonymous coding polymorphisms or for clinically-associated polymorphisms described in previous studies. The following SNPs were selected in this study: SLC2A1 -2841A>T (rs710218), VEGFA+936C>T (rs3025039) [NM_001025366.1:c.*237C>T], APEX1 Asp148Glu (T>G, rs1130409) [NM_001641.2:c.444T>G], HIF1A Pro582Ser (C>T, rs11549465) [NM_001530.2:c.1744C>T], and HIF1A Ala588Thr (G>A, rs11549467) [NM_001530.2:c.1762G>A]. Genotyping
The SNaPshot assay was performed according to the manufacturer’s instructions (ABI PRISM SNaPShot Multiplex kit; Applied Biosystems, Foster City, CA, USA). Briefly, the genomic DNA flanking the SNP of interest was amplified with the use of a PCR reaction with forward and reverse primer pairs and standard PCR reagents. The 10 μL reaction volume contained 10 ng of genomic DNA, 0.5 pM of each oligonucleotide primer, 1 mL Etofibrate of 10× PCR buffer, 250 μM dNTP (2.5 mM each), and 0.25 units www.selleckchem.com/products/tpx-0005.html i-StarTaq DNA Polymerase (5 units/μL; iNtRON Biotechnology, Sungnam, Kyungki-Do, Korea). PCR reactions were carried out as follows: 10 min at 95°C for 1 cycle, and 35 cycles at 95°C for 30 s, followed by 1 extension cycle at 72°C for 10 min. After amplification, the PCR products were treated with 1 U each of shrimp alkaline phosphatase (SAP) and exonuclease I (Roche Diagnostics, Mannheim, Germany) at
37°C for 75 min and 72°C for 15 min to purify the amplified products. One μL of the purified amplification products was added to a SNaPshot Multiplex Ready reaction mixture containing 0.15 pmol of genotyping primer for a primer extension reaction. The primer extension reaction was carried out for 25 cycles of 96°C for 10 sec, 50°C for 5 sec, and 60°C for 30 sec. The reaction products were treated with 1 U of SAP at 37°C for 1 hr and 72°C for 15 min to remove excess fluorescent dye terminators. One μL of the final reaction samples containing the extension products was added to 9 μL of Hi-Di formamide (Applied Biosystems). The mixture was incubated at 95°C for 5 min, followed by 5 min on ice, then the mixture was analyzed by electrophoresis on an ABI Prism 3730xl DNA analyzer. Analysis was carried out using Genemapper software (version 3.0; Applied Biosystems). Table 1 shows the primer sets and Tm used for the SNaPshot assay.