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2.2. RNA isolation and quantitative reverse transcription-polymerase chain reaction
Total RNA was extracted from the peripheral WBCs with TRIzol reagent (CWBIO, Beijing, China) and reverse-transcribed into cDNA using a cDNA synthesis kit (TransGen Biotech, Beijing, China) ac-cording to the manufacturer’s instructions. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed in a Light Cycler 480 system (Roche, Shanghai, China) with SYBR Green and the following gene-specific primers: GAPDH: 5′-ACTGGCGTCTTCACC ACCAT-3′ and 5′-GCAGGAGGCATTGCTGATGA-3′; IRF5: 5′-GGGAAAT ACACCGAAGGCG-3′ and 5′-TCCTGCACCAAAAGAGTAATCCT-3′; IL-6: 5′-CTGGCAGAAAACAACCTGAAC-3′ and 5′-ATGATTTTCACCAGGCAA GTC-3′; TNF-α: 5′-CATCTTCTCGAACCCCGAGT-3′ and 5′-GGATACCA CTCCCAACAGACC-3′; IP-10: 5′-AACTGTACGCTGTACCTGCAT-3′ and 5′-GCATCGATTTTGCTCCCCTC-3′; IL-10: 5′-GGCTTGTCACTCGGGGT TCG-3′ and 5′-GCCAAGCCTTGTCTGAGATGA-3′. The cycling conditions were 40 cycles of 95℃ for 15 s and 60℃ for 1 min. The relative tran-scription levels of the target genes were calculated via the 2− Ct method using GAPDH mRNA levels as in reference .
Genomic DNA was isolated from the peripheral WBCs using the BloodGen Mini kit (Genomic DNA Isolation Mini Kit; CW0540, CWBIO, Beijing, China). Genotyping for IRF5 rs77571059 and rs2004640 single nucleotide polymorphisms (SNPs) was performed by polymerase chain reaction (PCR) using the Verity 96-well PCR system (Applied Biosystems, Foster City, CA, USA). The identified PCR products were sequenced in a 3730XL sequencer (Applied Biosystems, Foster City, CA, USA) using the following primers: rs77571059: 5′-CATTCCAGATTGC CAAAAGAGC-3′ and 5′-CAACCTGCCGGGCATTC-3′; rs2004640: 5′-CAAGACGCGGAAGTGCCC-3′ and 5′-GCGAGTGCATCGAAAGTAA GGA-3′.
2.4. Blood processing and flow cytometry
Whole blood samples (30 μl each) were stained with PerCPCy5.5-anti-CD45, FITC-anti-CD14, and PE-anti-CD19 (BD Pharmingen, San Diego, CA, USA) for 30 min at room temperature in the dark and red blood SPDP were lysed. The WBCs were fixed, permeabilised with Transcription Factor Buﬀer (BD Biosciences, San Jose, CA, USA) for 50 min, stained with APC-labelled anti-IRF5 antibody (Novus, Littleton, CO, USA) for 50 min at 4 °C, and subjected to flow cytometry using the FACSCalibur platform (BD Biosciences). The data were analysed with FlowJo software (v.7.6.1) (Tree Star, Ashland, OR, USA).
2.5. Cytometric bead array (CBA) to measure plasma cytokine/chemokine levels
Plasma levels of TNF-α, IL-6, IL-10, and IP-10 were analysed using the CBA Flex Set system (BD Biosciences) according to the manu-facturer’s protocol. Flow cytometry was conducted using FACSCalibur (BD Biosciences). Quantitative analysis was performed with CellQuestPro and CBA software (BD Biosciences).
2.6. Western blotting
Total protein was extracted from paired samples of NSCLC and adjacent non-tumour lung tissues using the ProteinExt Mammalian Total Protein Extraction Kit (Transgen, Beijing, China) according to the manufacturer’s instructions; the protein concentration was measured via the bicinchoninic acid method. Total proteins (20 μg) were Lung Cancer 135 (2019) 47–55
separated by sodium dodecyl sulphate polyacrylamide gel electro-phoresis (SDS-PAGE) in 12% gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 2 h at room temperature with Tris-buﬀered saline containing 5% dried non-fat milk and incubated with primary anti-bodies against IRF5 (ab33478; Abcam, Cambridge, MA, USA) or β-actin (sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4℃. After washing with TBST (150 mM NaCl, 10 mM Tris-HCl [pH 8.0], and 0.5% Tween-20) three times for 10 min, the membranes were incubated with horseradish peroxidase-labelled goat anti-rabbit IgG (sc-2004; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature, then washed and incubated with electro-chemiluminescence agent for 1 min. Protein-specific signals were de-tected using X-ray films; the signal intensity was determined by den-sitometry using the Image Gauge V3.12 program (Fuji Photo Film, Tokyo, Japan) and expressed as the grey value.