4.13. Primer Design and Quantitative Real-Time PCR (qRT-PCR) Analysis

For each target gene monitored in qRT-PCR experiments (whose sequences were downloaded from the NCBI Gene database), primer sequences were designed using the Primer 3 Plus software by setting default parameters (https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi accessed on 22 June 2021). To exclude the formation of primer dimers and hairpins, primer sequences were bioinformatically screened with the IDT Oligo Analyzer tool (Integrated DNA Technologies, Inc., Coralville, Iowa, USA). Moreover, primer pairs specificity was evaluated by the in-silico PCR tool implemented in the UCSC Genome Browser. Primer pairs chosen are listed in Supplementary Table S6. The amplification by qRT-PCR was accomplished in a final volume (f.v.) of 5 μL per well, including 1 µL of EvaGreen qPCR Mix Plus (Solis BioDyne), 0.1 μL of forward primer (10 μM), 0.1 μL of reverse primer (10 μM), 8 ng of the qPCR cDNA template, and water to f.v. The reactions were performed in the CFX thermocycler (BioRad) with the following program: 10-min-denaturation at 95 °C, 40 cycles amplification (95 °C for 15 s; 60 °C for 20 s; 72 °C for 45 s, annealing and extension); 3 min at 72 °C and dissociation curve.

Six different genes (Actin; TATA binding protein (Tbp); RNA Polymerase II Subunit A, (Polr2a); Glyceraldehyde 3-phosphate dehydrogenase, (Gapdh); ribosomal protein lateral stalk subunit P0, (Rplp0); and ribosomal protein L32, (Rpl32)) were tested and categorized according to their coefficient of variation to choose the best reference genes for accurate normalisation of data (Supplementary Figure S4). Target-gene mRNA levels were quantified according to the ΔCt method and using the two best reference genes (Polr2a and Tbp; see Supplementary Figure S4) as housekeeping genes. Data were represented as expression value relative to the average expression of the gene among samples.