4.3. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Quantitative analysis of the mRNA expression of CCL18, CCR8 and PITPNM3 genes was performed by two-step reverse transcription PCR (RT-PCR). Total RNA was extracted from 50–100 mg tissue samples using an RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) and, for the in vitro study, from 300,000 cells using an RNeasy Mini Kit. cDNA was prepared from 1 μg of total cellular RNA in 20 μL of reaction volume using a FirstStrand cDNA synthesis kit and oligo-dT primers (Fermentas, Waltham, MA, USA). Quantitative assessment of mRNA levels was performed using an ABI 7500Fast real-time RT-PCR analyzer with Power SYBR Green PCR Master Mix reagent (Applied Biosystems, Waltham, MA, USA). Real-time conditions were 95 °C (15 s), 40 cycles at 95 °C (15 s), and 60 °C (1 min). According to melting point analysis, only one PCR product was amplified under these conditions. Each sample was analyzed in two technical replicates, and mean Ct values were used for further analysis. The relative quantity of a target, normalized to the levels of endogenous controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was calculated as the fold difference (2^dCt) and further processed using statistical analysis. Data were presented as tumor tissue absolute expression.

The GAPDH reference gene was selected because it is considered a suitable control in research on the expression of various genes in GBM [69]. The following primer pairs were used:

(5′-TCA TGG GTG TGA ACC ATG AGA A-3′ and 5′-GGC ATG GAC TGT GGT CAT GAG-3′) for GAPDH,

(5′-CTCTGCTGCCTCGTCTATACCT-3′ and 5′-CTTGGTTAGGAGGATGACACCT-3′) for CCL18,

(5′-GTGTGACAACAGTGACCGACT-3′ and 5′-CTTCTTGCAGACCACAAGGAC-3′) for CCR8,

(5′-TCGCTTGTCTCACCTGAAC-3′ and 5′-CAGGAACTCTCTGTAGACCTGG-3′) for PITPNM3.