TY - JOUR
T1 - Addressing the unsolved challenges in microRNA-based biomarker development
T2 - Suitable endogenous reference microRNAs for SARS-CoV-2 infection severity
AU - Belmonte, Thalia
AU - Perez-Pons, Manel
AU - Benítez, Iván D.
AU - Molinero, Marta
AU - García-Hidalgo, María C.
AU - Rodríguez-Muñoz, Carlos
AU - Gort-Paniello, Clara
AU - Moncusí-Moix, Anna
AU - Madè, Alisia
AU - Devaux, Yvan
AU - Martelli, Fabio
AU - Ortega, Alicia
AU - González, Jessica
AU - Torres, Gerard
AU - Barbé, Ferrán
AU - de Gonzalo-Calvo, David
N1 - Acknowledgments
Work supported by IRBLleida Biobank (B.000682) and Biobank and Biomodels Platform ISCIII PT23/00032. The human sample manipulation was performed in the Cell Culture Technical Scientific Service of the Universitat de Lleida (Lleida, Catalonia, Spain). The authors acknowledge all the COVID-19 patients and health workers involved in the study.
Copyright © 2024. Published by Elsevier B.V.
PY - 2024/6
Y1 - 2024/6
N2 - 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.
AB - 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.
KW - Biomarker
KW - COVID-19
KW - Endogenous controls
KW - MicroRNA
KW - Normalization
KW - Reference genes
UR - http://www.scopus.com/inward/record.url?scp=85192286780&partnerID=8YFLogxK
UR - https://pubmed.ncbi.nlm.nih.gov/38688344
U2 - 10.1016/j.ijbiomac.2024.131926
DO - 10.1016/j.ijbiomac.2024.131926
M3 - Article
C2 - 38688344
SN - 0141-8130
VL - 269
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
IS - Pt 2
M1 - 131926
ER -