2.3. RNA Extraction and Real-Time PCR

Total RNA extraction of each FFPE sample was performed from 5 or 6 sections (5 µm) using the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions and evaluated for RNA concentrations through a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) spectrophotometer.

The reverse transcription (RT) step was performed using the SuperScript™ IV VILO™ Master Mix (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), adding 250 ng of RNA and utilizing 5 μL of 1:5 diluted cDNA.

L1 and E6 EcPV2 genes were tested for their expression using specific primer sets and probed (Table 1) at the concentration of 200 nM, and 1 μM for each primer combination was added to the 25 μL PCR mixture at the final concentration of 1× master mix (iTaq Universal ProbsSupermix, Bio-Rad, Irvine, CA, USA) with the following thermal profile: 95 °C for 10′, then 39 cycles of 95 °C for 15′′ and 60 °C for 60′′ in a CFX96™ Real-Time System. RNA was used as the control to exclude possible contaminations by EcPV2 genomic DNA.

PD-1 and PD-L1 were selected to test their related gene expression. Primers for the PD-1 gene (PDCD1) and PD-L1 gene (CD274) were designed through Primer3web tool v. 4.1.0 [31] and reported in Table 1. B2M gene expression was used to normalize the host gene expression [32] utilizing primers previously reported (Table 1). The amplification was conducted using the SsoFast EvaGreen Supermix (BioRad, Hercules, CA, USA) in a CFX96 Real-Time System with 5 μL of 1:5 diluted cDNA. Each sample was tested in triplicates and fluorescence data were collected at the end of the second step of each cycle. A threshold cycle of 38 was set as the cut-off for positivity and samples with Cq > 38 were considered negative. All PCR products including the positive control were subjected to direct sequencing.