*2.3. E6/E7 mRNA HPV Detection*

E6/E7 mRNA of the most prevalent HPVs was tested in the cervical samples positive for the most prevalent HR HPVs DNA and HR HPV DNA negative samples. The HR-HPV-negative samples were included in E6/E7 mRNA testing because the study aimed to determine the mRNA test's clinical characteristics by evaluating and comparing it with the HPV DNA test. Total RNA was extracted from the prepared sample using the miRNeasy Mini Kit and QIAcube robotic workstation (Qiagen, Hilden, Germany) following the manufacturer's instructions. The total RNA was eluted in 50 µL ultrapure water free from nucleases. Following the manufacturer's recommendations, potentially present contaminants were removed using the TURBO DNA-free Kit (Invitrogen/ThermoFisher Scientific, Waltham, MA, USA). The routine procedure for removing contaminants using the kit above included the addition of 5 µL of 10× TURBO DNase Buffer and 1 µL of TURBO DNase enzyme into each sample of extracted total RNA, with incubation for 30 min at a temperature of 37 ◦C. After the action of the enzyme, 5 µL of inactivation reagent (Dnase Inactivation Reagent) was added, with incubation for 5 min, at room temperature and occasional vortexing. After that, centrifugation was performed (90 s, 10,000× *g*). The supernatant was carefully transferred to a nuclease-free tube. The real-time reverse transcription PCR (RT-PCR) analysis, marked as the HPV mRNA test, was performed using specific primers and TaqMan probes to detect the E6/E7 mRNA of individual HPV

genotypes. The sequences for the primers and probes (Table 1) were adopted from Lindh et al. (2007) [19] and purchased from Life Technologies (Carlsbad, CA, USA). The AgPath-ID One-Step RT-PCR Kit (Applied Biosystems, Waltham, MA, USA) was used for the real-time RT-PCR. A separate reaction mixture was prepared for each set of primers and TaqMan probes. The reaction was prepared to a final volume of 25 µL containing: 12.5 µL 2× RT-PCR Buffer, 1 µL of 25× RT-PCR Enzyme Mix, primers to a final concentration of 300 nM, the probe to a final concentration of 200 nM, 1 µL of RNase Inhibitor reagent (Applied Biosystems, Waltham, MA, USA), 5 µL of isolated total RNA, and DEPC-treated nuclease-free water (Invitrogen, Waltham, MA, USA). Real-time PCR was performed on the Applied Biosystems 7500 Real-Time PCR Systems (ThermoFisher Scientific, Waltham, MA, USA). After the reverse transcription reaction at 48 ◦C for 30 min, the inactivation of reverse transcriptase and the activation of Taq polymerase were performed at 95 ◦C for 10 min. After that, 45 cycles of PCR amplification were carried out with denaturation at 95 ◦C for 15 s and annealing and elongation at 58 ◦C for 1 min. The data were analyzed with the Applied Biosystems Software v2.0.6 (ThermoFisher Scientific, Waltham, MA, USA) and the GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).

**Table 1.** Primer and probe sequences used for the RT-PCR analysis.


F—forward; R—reverse; P—TaqMan probe; \*—antisense; FAM—6-carboxyfluorescein; TAMRA—6-carboxyte tramethylrhodamine.
