Identification and validation of circulating miRNAs as endogenous controls in obstructive sleep apnea

by Fernando Santamaria-Martos, Ivan Benítez, Andrea Zapater, Cristina Girón, Lucía Pinilla, Jose Manuel Fernandez-Real, Ferran Barbé, Francisco Jose Ortega, Manuel Sánchez-de-la-Torre

microRNAs (miRNAs) are non-coding RNAs highly relevant as biomarkers for disease. A seminal study that explored the role of miRNAs in obstructive sleep apnea syndrome (OSA) demonstrated their usefulness in clinical management. Nevertheless, the miRNAs that may act as endogenous controls (ECs) have not yet been established. The identification of ECs would contribute to the standardization of these biomarkers in OSA. The objective of the study is to identify miRNAs that can be used as ECs in OSA. We evaluated 100 patients divided into two different cohorts: a learning cohort of 10 non-OSA and 30 OSA patients, and a validation cohort (20 non-OSA and 40 OSA patients). In the learning cohort, a profile of 188 miRNAs was determined in plasma by TaqMan Low Density Array. The best EC candidates were identified by mean center+SD normalization and concordance correlation restricted normalization. The results were validated using NormFinder and geNorm to assess the stability of those ECs. Eight miRNAs were identified as EC candidates. The combination miRNA-106a/miRNA-186 was identified as the most stable among all candidates. We identified a set of ECs to be used in the determination of circulating miRNA in OSA that may contribute to the homogeneity of results.