title: MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA.
abstract: MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundancein vitro. The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples.
authors.affiliation: Division of Cardiology, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Amanda Marie
authors.initials: AM
authors.lastname: James
authors.affiliation: Division of Cardiology, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Meredith B
authors.initials: MB
authors.lastname: Baker
authors.affiliation: Department of Biomedical Engineering, Rice University, Houston, TX, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
authors.affiliation: Division of Cardiology, Emory University School of Medicine, Atlanta, GA, USA;; Atlanta Veterans Administration Medical Center, Decatur, GA, USA.
authors.forename: Charles D
authors.initials: CD
authors.lastname: Searles
mesh_terms: D035683:MicroRNAs / Q000032:analysis*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D035683:MicroRNAs
keywords: PCR.; cardiovascular disease; molecular beacon, microRNA, microRNA detection