*2.6. Real-Time Quantitative and Semi-Quantitative Polymerase Chain Reaction*

All qRT-PCR and semi-qRT-PCR reactions were performed using a BioRad C1000 Touch Thermal Cycler (Biorad, Hercules, CA, USA) in 96-well qPCR plates. All qRT-PCR reactions were carried out in triplicate wells per plate using three separate plates and average cycle threshold (Ct) values were used for quantification. Relative expression was calculated applying the ΔCt method for iSGECs, SGCLs, and Hela cells normalized to the expression of GAPDH. Per well, 10 μL of RT2 SYBR Green Fast Master Mix (Qiagen, Valencia, CA, USA) was added to 2 μL of cDNA, 1 μL of forward and reverse primers (10 mM) (IDT, Newark, NJ, USA) and 7 μL of nuclease-free water. Primers are listed in Supplementary Table S1 along with their respective target for cell expression characterization purposes.

Semi-qRT-PCR was employed to detect the expression of the SV40Lt transcript at multiple passage numbers (Supplementary Figure S1). Products from semi-qRT-PCR reactions were run on a 1% agarose TAE gel alongside a 1 kb DNA ladder (New England Biolabs, Ipswich, MA, USA) for 35 min and visualized using a gel doc system (GE Healthcare Life Sciences, Chicago, IL, USA).
