Identifying potential biomarkers for mild COVID-19 in saliva by miRNA profiling using NGS and ddPCR

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression by targeting messenger RNA (mRNA). Due to this regulatory function, viruses often modulate host miRNA expression to facilitate their own replication. Conversely, host cells regulate miRNA expression to activate the immune system response. As a result, miRNAs represent promising biomarkers for infectious diseases, including viral respiratory infections, in which the upper airways serve as the primary entry point. Therefore, saliva provides valuable insights into the patient's condition. Advancing techniques and methodologies for saliva analysis is essential. In the present study, saliva screening for miRNA expression was performed using next generation sequencing (NGS). We then validated our findings with digital droplet PCR (ddPCR) and quantitative PCR (qPCR). Additionally, we conducted in-silico analyses to identify the target genes and biological pathways associated with the dysregulated miRNAs and their roles in SARS-CoV-2 infection. The screening step pointed to miR-590-3p and miR-374a-5p being downregulated in COVID-19. Subsequent validation using qPCR and droplet digital PCR (ddPCR) confirmed the downregulation of these miRNAs in COVID-19 cases. Under the experimental conditions evaluated in this study, ddPCR showed improved analytical sensitivity and quantitative agreement compared with RT-qPCR for detecting low-abundance salivary miRNAs, thereby strengthening the robustness of our findings. Under the experimental conditions evaluated in this study, ddPCR showed improved analytical sensitivity and quantitative agreement compared with RT-qPCR for the detection of low-abundance salivary miRNAs. The functional analyses revealed that both miRNAs are involved in viral processes and apoptosis regulation. Moreover, the conservation of miR-374a-5p and miR-590-3p across different groups of mammals highlights the relevance of their regulatory roles. Taken together, these results suggest that miR-374a-5p and miR-590-3p may serve as suitable biomarkers for SARS-CoV-2 infection.