Total RNA was extracted from lung samples (cancer tissue obtained from the center of lung lesion and macroscopically unchanged lung tissue obtained from the most distant site from the resected lesion) using Universal RNA Purification Kit (Eurx, Gdansk, Poland) according to the manufacturer’s recommendations. The qualitative and quantitative assessments of RNA samples were determined by minielectrophoresis in polyacrylamide gel using RNA 6000 Pico/Nano LabChip kit (Agilent 2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA).

Complementary DNA (cDNA) was transcribed from 100 ng of total RNA, using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA) in a total volume of 20 μl per reaction. Reverse transcription (RT) master mix contained: 10× RT buffer, 25× dNTP Mix (100 mM), 10× RT Random Primers, MultiScribe™ Reverse Transcriptase, RNase Inhibitor and nuclease-free water. RT reaction was performed in a Personal Thermocycler (Eppendorf, Hamburg, Germany) in the following conditions: 10 min at 25 °C, followed by 120 min at 37 °C, then the samples were heated to 85 °C for 5 s, and hold at 4 °C.

The relative expression of the FAM107A gene was assessed in qPCR reactions using Micro Fluidic Cards with pre-loaded selected assays: Hs00200376_m1 for the FAM107A (family with sequence similarity 107A) gene and Hs00382667_m1 for ESD (esterase D) as the reference gene. The PCR mixture contained: 50 μl cDNA (50 ng) and 50 μl TaqMan® Universal Master Mix (Applied Biosystems, Carlsbad, CA). TaqMan Array card was centrifuged twice for 1 min at 1,200 rpm to fill the wells with PCR mixture. Then, it was sealed and placed in a 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA). The PCR conditions were as follows: after initial incubation at 50 °C for 2 min and AmpliTaq Gold® DNA polymerase activation at 94.5 °C for 10 min, real-time PCR amplification was processed in 40 cycles of 30 s denaturation at 97 °C, followed by 1 min elongation step at 59.7 °C.

The relative expression of FAM107A in the studied samples was assessed using the comparative delta-delta C_T method (TaqMan Relative Quantification Assay software, Applied Biosystems, Carlsbad, CA) and presented as RQ value, adjusted to ESD expression level. RNA isolated from normal lung tissue (Human Lung Total RNA, Ambion®, Life Technologies, Carlsbad, CA) served as a calibrator sample. RNAs obtained from macroscopically unchanged lung tissues formed the control group.

The extraction of genomic DNA from NSCLC specimens was performed using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The quality and quantity of isolated DNA was spectrophotometrically assessed (Eppendorf BioPhotometer™ plus, Eppendorf, Hamburg, Germany). DNA samples with a 260/280 nm ratio in the range 1.8–2.0 were considered as high quality and used in further analysis.

Methylation status of the FAM107A gene was assessed by methylation-specific polymerase chain reaction (MSP) using bisulfite converted DNA. Genomic DNA (1 μg) was modified with sodium bisulfite, using the CpGenomeTM Turbo Bisulfide Modification Kit (CHEMICON International, Millipore, Temecula, CA), according to the manufacturer’s protocol. Concentration and purity of the modified DNA was spectrophotometrically estimated at 260/280 nm in a biophotometer (Eppendorf BioPhotometer™ plus, Eppendorf, Hamburg, Germany). The conventional MSP method was performed according to Herman et al. (1996), with some modifications. Briefly, MSP was performed for each sodium bisulfite modified DNA sample using AmpliTaq Gold® 360 DNA Polymerase (Applied Biosystems, Carlsbad, CA). Amplifications were conducted in a total volume of 12.5 μl in a Thermocycler SureCycler 8800 (Agilent Technologies, Santa Clara, CA). MSP master mix contained: 1,000 ng DNA, 0.7 μM of each primer (Sigma-Aldrich, Poznan, Poland), 2.5 μM dNTPs mix, 2.5 μM MgCl₂, Hot Start AmpliTaq Gold® 360 Polymerase (5 U/μl), 10x Universal PCR buffer and nuclease-free water. PCR conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles involving denaturation at 95 °C for 45 s, annealing temperature – appropriate for a given primer (see Table 2) – for 45 s and elongation at 72 °C for 1 min; the final elongation step was done at 72 °C for 10 min.

The set of primers for the studied gene was flanking the 1 kb 5′ region upstream from the translation start point. Primers for the methylation-specific PCR were designed according to the criteria described by Feltus et al. (2003). Primer sequences for the methylated and unmethylated FAM107A promoter regions are given in Table 2.

In each PCR reaction, positive and negative MSP controls were included. CpGenome Universal Methylated DNA (enzymatically methylated human male genomic DNA) served as a positive methylation control and CpGenome Universal Unmethylated DNA (human fetal cell line) was used as a negative control (Chemicon International, Millipore, Temecula, CA). Additionally, blank samples with nuclease-free water were used instead of DNA as a control for PCR contamination.

The MSP products were separated electrophoretically on 2 % agarose gel and their concentration (ng) of MSP products (U and M DNA alleles) was estimated spectrophotometrically, using DNA1000 LabChip Kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Afterwards, the Methylation Index (MI) was assessed for each sample, using the following formula: peak height of methylated products/(peak height of methylated products +peak height of unmethylated product), MI = (M)/(M + U).

The Kruskal-Wallis and Mann-Whitney U tests were used to compare the levels of relative expression (RQ values) between NSCLC subtypes, i.e., SCC, AC, and LCC, shown in box and whisker plots. Spearman’s rank correlation coefficient, Mann-Whitney U test, and Kruskal-Wallis test were performed in order to evaluate the relationship between the expression level of the studied gene and examined parameters (patients’ characteristics: age, gender, history of smoking and tumor staging according to pTNM and AJCC classifications). The results of relative expression analysis (RQ values) are presented as means ± SE and means ± SD. The accepted level of statistical significance was estimated at P < 0.05. Statistica for Windows 10.0 program (StatSoft, Cracow, Poland) was applied for calculations.