Abstract
BackgroundMicroRNAs (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.MethodsWe 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 Aβ42 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.ResultsNine 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.ConclusionsWe 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.
Highlights
MicroRNAs are small noncoding RNA molecules that regulate the activity of specific messenger RNA targets by binding to their 3′-untranslated regions (UTRs)
Patient characteristics The screening study consisted of 19 females with a diagnosis of mild to moderate Alzheimer’s disease (AD) with confirmed pathological levels of Amyloid-β42 protein (Aβ42) in Cerebrospinal fluid (CSF) (≤ 600 pg/ml) and Mini-Mental State Examination (MMSE) score ≥ 20 (Table 1)
The stability assessment cohort consisted of 126 individuals as follows: subjects without AD or any kind of dementia and negative for core CSF biomarkers of AD, subjects with mild cognitive impairment (MCI) and negative for core CSF biomarkers (MCI −), 22 subjects with MCI and positive for CSF biomarkers (MCI +), and 55 subjects with AD and positive for CSF biomarkers
Summary
MicroRNAs (miRNAs) are small (typically 22 nt in size) noncoding RNA molecules that regulate the activity of specific messenger RNA (mRNA) targets by binding to their 3′-untranslated regions (UTRs) This interaction suppresses the translation of the mRNA or induces its degradation [1]. Some experimentally induced artifacts, e.g., starting sample amount, collection and storage conditions, and miRNA extraction/transcription efficiency, profoundly affect the final result of RT-qPCR, eventually affecting the interpretation of the biological response. To overcome this problem, the use of endogenous miRNAs as normalizers is the method of choice because their expression is affected by the same variables as the expression of target genes [7].
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