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

Thalia Belmonte, Manel Perez-Pons, Iván D. Benítez, Marta Molinero, María C. García-Hidalgo, Carlos Rodríguez-Muñoz, Clara Gort-Paniello, Anna Moncusí-Moix, Alisia Madè, Yvan Devaux, Fabio Martelli, Alicia Ortega, Jessica González, Gerard Torres, Ferrán Barbé, David de Gonzalo-Calvo*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

Original languageEnglish
Article number131926
JournalInternational Journal of Biological Macromolecules
Volume269
Issue numberPt 2
Early online date28 Apr 2024
DOIs
Publication statusE-pub ahead of print - 28 Apr 2024

Keywords

  • Biomarker
  • COVID-19
  • Endogenous controls
  • MicroRNA
  • Normalization
  • Reference genes

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