Qtower3 link

The RNA was transcribed into cDNA (SuperScript III RT, Thermo Fisher Scientific, USA). Basing upon the measurement after RNA purification, the final concentration was 25 ng/µL. All steps from RNA isolation to cDNA synthesis were performed in parallel for all samples of each experiment in order to avoid experimental variations.
RT-qPCR was performed in technical duplicates using 2.5 ng/µL cDNA in each reaction and a primer concentration of 0.5 µM. The qTower^{3 (link)} (Analytik Jena, Germany), High Green Mastermix (Thermo Fisher Scientific, USA), qPCR-Soft 3 (Analytik Jena, Germany) and self-designed intron spanning primers were used (Eurofins, Luxembourg). Primers were designed by using Primer-BLAST (NCBI, USA) followed by a PCR-Check (Eurofins Oligo Analyse Tool, Luxembourg) to ensure in silico qPCR specificity. Our criteria were length ca 20 bp, annealing temperature 60 °C, max product length 200 bp, intron spanning, covering possible transcript variants. The RT-qPCR protocol included an initial step of 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 95 °C/15 s, 60 °C/30 s and 72 °C/30 s. A step of 95 °C for 15 s forms the transition to melting curve analysis (60–95 °C).

Gene expression was estimated measuring the mRNA from cell extraction by RT-qPCR with qTOWER (3 (link)) (Analytik Jena, Germany), using One-step NZYSpeedy RT-qPCR Probe Kit, ROX (NZYTech, Lisbon, Portugal). 10 ng/µl RNA was employed, and the threshold cycle (CT) values from each biological assay were plotted with two experimental replicates following the manufacturer’s procedure. Melting curve analysis was used to monitor the specificity of primers and probes. Results were normalized to the GAPDH housekeeping gene, and gene relative expression was employed by the ΔC_T expression/ΔC_T negative control ratio.