Gel doctm imager

At 3 h from irradiation, total RNA was extracted using Trizol^® Reagent (Sigma-Aldrich) according to the manufacturer’s instructions and quantified using NANODROP 2000 (Thermo Fisher Scientific). Primers were obtained from Invitrogen, and their sequences are reported in Table 1. β-actin was chosen as the housekeeping gene. RT-PCR was carried out through the Qiagen^® One Step RT-PCR Kit (Qiagen, Crawley, UK) according to the manufacturer’s protocols and using total RNA at a concentration of 50 ng/reaction for each sample. The thermal cycling program consisted of 50 °C for 30 min (reverse transcription), 95 °C for 15 min (DNA-polymerase activation), 39 two-step cycles of 94 °C for 1 min (denaturation), 57 °C for 1 min (annealing), and 72 °C for 1 min (elongation). The procedure was carried out using the iCycler iQ™ (Bio-Rad). The PCR products were separated by 2% agarose gel electrophoresis and visualised by Gel Red Nucleic Acid staining at 1:10,000 (Biotium, Hayward, CA, USA). The images of the gel were captured with Gel Doc^TM Imager (Bio-Rad) and analysed with Image Lab software (Bio-Rad). To obtain a semiquantitative assessment of the gene expression, data were expressed as normalised ratios by comparing the integrated density values for target genes with those for β-actin.

At 7 days from seeding, total RNA was extracted using TriReagent according to the manufacturer’s instructions and quantified using NANODROP 2000 (Thermo Scientific, Carlsbad, CA, USA). Primers’ sequences are reported in Table 1. GAPDH was chosen as the housekeeping gene. RT-PCR was carried out through the qPCRBIO SyGreen 1-Step Go Lo-ROX according to the manufacturer’s protocol and using total RNA at a concentration of 75 ng/reaction for each sample. The thermal cycling program consisted of 50 °C for 10 min (reverse transcription), 95 °C for 2 min (DNA-polymerase activation), 40 two-step cycles of 95 °C for 5 s (denaturation), and 62 °C for 25 s (annealing and elongation). The procedure was carried out using the C1000 Touch thermal cycler (Bio Rad, Hercules, CA, USA). The PCR products were separated by 1% agarose gel electrophoresis and visualized by Gel Red Nucleic Acid staining at 1:10,000. The images of the gel were captured with Gel Doc^TM Imager (Bio-Rad) and analyzed with Image Lab software (Bio-Rad). To obtain a semiquantitative assessment of gene expression, normalized ratios were obtained by comparing the integrated density values for target genes with those of GAPDH. Then, results were expressed as a percentage with respect to the cultures grown on tissue-culture polystyrene plates considered as 100%.