Materials and methods

Experiments were carried out on an HaCaT cell; an immortal human keratinocyte cell line. Cells were cultivated in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (Gibco®), and gentamicin 10 mg/ml. Cells were grown in an incubator at 370°C with 5% CO2 supply, and fresh complete medium was changed every 2–3 days. For passaging, the monolayer of adherent cells was incubated with 0.25% Trypsin–Versene® for 10 minutes at 37°C. Storage cells (in the medium supplied with 10% dimethyl sulfoxide; DMSO) were snap-frozen in liquid nitrogen.

For the experiment, cells were seeded onto tissue culture flasks at a density of 1 x 106 cells per 90 mm dish containing 5 ml of complete medium. The culture medium was changed when cells reached monolayer the day before the experiment. Treatment of the cells was carried out in aseptic conditions by low intensity  irradiation (1270 nm, exposition time 1 and 5 minutes, distance 2–3 cm).

Two hours later cells were rinsed with phosphate buffered saline (PBS) and lysed with buffer RLT for ribonucleic acid (RNA) isolation. RNA isolation was carried out according to the following protocol.

Cells were lysed using 300 μl buffer RLT with β-mercaptoethanol and immediately homogenised. Then 590 μl ddH2O with 10 μl proteinase K (10 mg/ml) were added to the samples and mixed by pipetting. After a 10-minute incubation period at 55°C, the samples were centrifuged at 10000 xg for 5 minutes at room temperature, and the supernatants were transferred to a new tube. Afterward, 450 μl of ethanol was added to each sample of the homogenised lysate, the samples, including any precipitate, were then transferred to an RNeasy mini spin column placed in a 2 ml tube. The samples were centrifuged for 15 seconds at 8000 xg twice. In order to wash the spin column membrane, 350 μl of buffer RW1 were added to the samples with consequent centrifugation. The samples were treated with DNase in buffer RDD for 15 minutes at room temperature. The columns were rinsed with 350 μl of buffer RW1 and twice with 500 μl of buffer RPE. RNA was eluted from the column with 30–50 μl of RNase-free water. For the next step of the analysis, complementary DNA (cDNA) was synthesised from the mRNA samples using the reverse transcription.

The RNA concentration was measured using NanoDrop 1000 Spectrophotometer (Thermo Scientific, United States), after which the sample concentrations were equalised in ddH2O.

Reverse transcription was performed in PCR tubes in a volume of 200 μL, containing a buffer, deoxyribonucleotide triphosphate (dNTP), 100 U M-MLV reverse transcriptase, 20 U RNasin, RNase inhibitor, 500 ng Oligo(dT) primers, and RNA to the final concentration not more than 100 ng/μL. The reaction mixture was incubated for 1 hour at 37°C.

Real-time PCR (qPCR) was performed using oligonucleotide probes with fluorescent labels and 2.5× reaction mixture with a ROX reference dye. Primers and probes were synthesised by DNA synthesis.

Amplification was performed using a PCR machine (Bio-Rad, iQ4) using the following programme:

  • Denaturation at 95°C, 4 minutes
  • Denaturation at 95°C, 15 seconds
  • Annealing at 58°C, 15 seconds
  • Elongation at 72°C, 15 seconds
  • Steps 2–4 were repeated 35 times.

The expression of target genes was normalised using the housekeeping glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The amplification of the GAPDH gene and the examined genes were conducted in separate tubes. Results were calculated using the  2–ΔΔ Ct method, which shows the fold change in gene expression in the treated compared to untreated cells14. ΔΔCt was calculated as follows:

ΔΔCt = ΔCt (treated cells)  —  ΔCt (untreated cells)

Each value was calculated thus:

ΔCt = Ct (the analysed gene) — Ct (GAPDH).

Each sample was analysed in triplicate.