Identification and validation of endogenous control miRNAs in plasma samples for normalization of qPCR data for Alzheimer's disease.

BACKGROUND: MicroRNAs (miRNAs) are noncoding RNAs that are highly relevant as disease biomarkers. Several studies that explored the role of miRNAs in Alzheimer's disease (AD) demonstrated their usefulness in clinical identification. Nevertheless, 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 AD. The objective of the study was to identify miRNAs that can be used as ECs in AD. METHODS: We evaluated 145 patients divided into two different cohorts. One was a discovery cohort of 19 women diagnosed with mild to moderate AD (Mini-Mental State Examination (MMSE) score ≥ 20) and with confirmed pathologic levels of Aß42 in CSF. The stability assessment cohort consisted of 126 individuals: 24 subjects without AD or any kind of dementia and negative for all core CSF biomarkers of AD, 25 subjects with MCI and negative for CSF biomarkers (MCI -), 22 subjects with MCI and positive for CSF biomarkers (MCI +), and 55 subjects with AD and positive for CSF biomarkers. In the discovery cohort, a profile of 384 miRNAs was determined in the plasma by TaqMan low-density array. The best EC candidates were identified by mean-centering and concordance correlation restricted normalization methods. The stability of the EC candidates was assessed using the GeNorm, BestKeeper, and NormFinder algorithms. RESULTS: Nine miRNAs (hsa-miR-324-5p, hsa-miR-22-5p, hsa-miR-103a-2-5p, hsa-miR-362-5p, hsa-miR-425-3p, hsa-miR-423-5p, hsa-let-7i-3p, hsa-miR-532-5p, and hsa-miR-1301-3p) were identified as EC candidates in the discovery cohort. The validation results indicated that hsa-miR-103a-2-5p was the best EC, followed by hsa-miR-22-5p, hsa-miR-1301-3p, and hsa-miR-425-3p, which had similar stability values in all three algorithms. CONCLUSIONS: We identified a profile of four miRNAs as potential plasma ECs to be used for normalization of miRNA expression data in studies of subjects with cognitive impairment.

Circulating MicroRNA Profile Associated with Obstructive Sleep Apnea in Alzheimer's Disease.

The diagnosis of obstructive sleep apnea (OSA) in Alzheimer's disease (AD) by polysomnography (PSG) is challenging due to the required collaboration of the patients. In addition, screening questionnaires have demonstrated limited usefulness with this subpopulation. Considering this, we investigated the circulating microRNA (miRNA) profile associated with OSA in AD patients. This study included a carefully selected cohort of females with mild-moderate AD confirmed by biological evaluation (n = 29). The individuals were submitted to one-night PSG to diagnose OSA (apnea-hypopnea index ≥ 15/h) and the blood was collected in the following morning. The plasma miRNA profile was evaluated using RT-qPCR. The patients had a mean (SD) age of 75.8 (5.99) years old with a body mass index of 28.6 (3.83) kg m-2. We observed a subset of 15 miRNAs differentially expressed between OSA and non-OSA patients, of which 10 were significantly correlated with the severity of OSA. Based on this, we built a prediction model that generated an AUC (95% CI) of 0.95 (0.88-1.00) including 5 of the differentially expressed miRNAs that correlated with OSA severity: miR-26a-5p, miR-30a-3p, miR-374a-5p, miR-377-3p, and miR-545-3p. Our preliminary results suggest a plasma miRNA signature associated with the presence of OSA in AD patients. Further studies will be necessary to validate these findings.