Addressing the unsolved challenges in microRNA-based biomarker development: Suitable endogenous reference microRNAs for SARS-CoV-2 infection severity

• Published in: International journal of biological macromolecules 2024-06, Vol.269 (Pt 2), p.131926-131926, Article 131926
• Main Authors: Belmonte, Thalia, Perez-Pons, Manel, Benítez, Iván D., Molinero, Marta, García-Hidalgo, María C., Rodríguez-Muñoz, Carlos, Gort-Paniello, Clara, Moncusí-Moix, Anna, Madè, Alisia, Devaux, Yvan, Martelli, Fabio, Ortega, Alicia, González, Jessica, Torres, Gerard, Barbé, Ferran, de Gonzalo-Calvo, David
• Format: Article
• Language: English
• Subjects: Adult; Aged; Biomarker; biomarkers; Biomarkers - blood; Circulating MicroRNA - blood; Circulating MicroRNA - genetics; COVID-19; COVID-19 - diagnosis; COVID-19 - genetics; COVID-19 infection; data collection; decision making; Endogenous controls; Female; hospitals; Humans; Male; MicroRNA; MicroRNAs - blood; MicroRNAs - genetics; Middle Aged; Normalization; Reference genes; Reproducibility of Results; SARS-CoV-2 - genetics; sequence analysis; Severe acute respiratory syndrome coronavirus 2; Severity of Illness Index
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2024.131926

Circulating cell-free microRNAs (miRNAs) are promising biomarkers for medical decision-making. Suitable endogenous controls are essential to ensure reproducibility. We aimed to identify and validate endogenous reference miRNAs for qPCR data normalization in samples from SARS-CoV-2-infected hospitalized patients. We used plasma samples (n = 170) from COVID-19 patients collected at hospital admission (COVID-Ponent project, www.clinicaltrials.gov/NCT04824677). First, 179 miRNAs were profiled using RT–qPCR. After stability assessment, candidates were validated using the same methodology. miRNA stability was analyzed using the geNorm, NormFinder and BestKeeper algorithms. Stability was further evaluated using an RNA-seq dataset derived from COVID-19 hospitalized patients, along with plasma samples from patients with critical COVID-19 profiled using RT-qPCR. In the screening phase, after strict control of expression levels, stability assessment selected eleven candidates (miR-17-5p, miR-20a-5p, miR-30e-5p, miR-106a-5p, miR-151a-5p, miR-185-5p, miR-191-5p, miR-423-3p, miR-425-5p, miR-484 and miR-625-5p). In the validation phase, all algorithms identified miR-106a-5p and miR-484 as top endogenous controls. No association was observed between these miRNAs and clinical or sociodemographic characteristics. Both miRNAs were stably detected and showed low variability in the additional analyses. In conclusion, a 2-miRNA panel composed of miR-106a-5p and miR-484 constitutes a first-line normalizer for miRNA-based biomarker development using qPCR in hospitalized patients infected with SARS-CoV-2.