Forward and reverse primer

An amount of 1 µg total RNA was transcribed using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) for complementary DNA (cDNA) synthesis. The qRT-PCR measurements were performed with reaction mixtures containing 5 µL LightCycler 480 SYBR Green I Master reaction mixture (Roche, Basel, Switzerland), 0.25 µL 25 µM forward and reverse primer (Biomers, Ulm, Germany), 2 µL water, and 2.5 µL 1:10 diluted cDNA. Primer sequences are listed in Table 2. Amplification and detection were accomplished on a LightCycler 480 Instrument II system (Roche, Basel, Switzerland). The PCR program included initial incubation for 5 min at 95 °C, 45 cycles of denaturation (95 °C, 10 s), annealing (specific annealing temperature, 15 s), and elongation (72 °C, 20 s). Following amplification, a melting curve analysis was performed. Relative mRNA gene expression was normalized on the relative succinate dehydrogenase complex flavoprotein subunit A (SDHA), hydroxymethylbilane synthase (HMBS), and ribosomal protein L13 (RPL13) gene expression was calculated based on the delta-delta Ct method considering PCR efficiency and internal calibration. Each condition was measured in biological and technical triplicates.

A total of 1 µg RNA was transcribed into cDNA by using the SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, San Diego, CA, USA), as described by the manufacturer. Regarding all gene expression measurements, 2.5 µL cDNA (1:10 diluted in water), 0.25 µL forward and reverse primer (Biomers, Ulm, Germany) with a final concentration of 25 µM, 2 µL water and 5 µL LightCycler 480 SYBR Green I Master reaction mixture (Roche, Penzberg, Germany) were used. The protocol for quantitative real-time PCR (qPCR) is shown in Table 2. The mRNA expression of the genes ß-actin (ACTB), glyceraldehyde-3-phosphatase-dehydrogenase (GAPDH) and ß2-microglobulin (ß2M) were used for normalization. Finally, a melting curve was measured after amplification for the analysis of purity. Three technical replicates were performed for each biological sample. All measurements were carried out by using a LightCycler480 (Roche, Penzberg, Germany). The ΔΔCt method, considering PCR efficiency, was used for the calculation of relative mRNA expression. All sequences of the primer used for qPCR are listed in Table S1.

For cDNA synthesis, 1 µg RNA was transcribed into cDNA using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). For p27, p53, p21, LMNB1, IL6, MCP1 and ICAM1 2.5 μL cDNA (1:10), 0.25 μL forward and reverse primer (Biomers, Ulm, Germany), 2.0 μL water and 5.0 μL LightCycler 480 SYBR Green I Master reaction mixture (Roche, Penzberg, Germany) was mixed for each measurement. After an initial incubation for 5 min at 95 °C, the qPCR protocol involves 45 cycles of denaturation (95 °C, 10 s), annealing (specific annealing temperature, 15 s) and elongation (72 °C, 20 s). Relative mRNA expression of measured targets was normalized to relative β-actin (β-ACTIN), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β2-microglobulin (β2M) mRNA expression. After amplification, melting curve analysis was performed. Measurements of gene expression were conducted using LightCycler 480 (Roche, Penzberg, Germany). Relative mRNA expression was calculated using the delta-delta Ct method considering PCR efficiency. Technical triplicates were done for each biological sample. Sequences of forward and reverse primers can be found in Table 3.

SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, San Diego, CA, USA) was used to transcribe 1 µg RNA into cDNA. For all gene expression measurements, 2.5 μL cDNA (1:10), 0.25 μL forward and reverse primer (Biomers, Ulm, Germany) with a final concentration of 25 µM (except IL6: 20 µM), 2.0 μL water and 5.0 μL LightCycler 480 SYBR Green I Master reaction mixture (Roche, Penzberg, Germany) were mixed. The qPCR protocol involves an initial incubation for 5 min at 95 °C and 45 subsequent cycles of denaturation (95 °C, 10 s), annealing (specific annealing temperature, 15 s) and elongation (72 °C, 20 s). β-actin (ACTB), glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) and β2-microglobulin (β2M) mRNA expression was used for normalization. Melting curve analysis was done after amplification and technical triplicates were performed for each biological sample. Measurements were carried out using a LightCycler 480 (Roche, Penzberg, Germany). Relative mRNA expression was calculated using the delta delta Ct method considering PCR efficiency. Sequences of the primers used for qPCR are listed in Table 2.