James Amanda Marie, Baker Meredith B, Bao Gang, Searles Charles D
Division of Cardiology, Emory University School of Medicine, Atlanta, GA, USA.
Department of Biomedical Engineering, Rice University, Houston, TX, USA.
Theranostics. 2017 Jan 15;7(3):634-646. doi: 10.7150/thno.16840. eCollection 2017.
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) abundance . 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.
微小RNA（miRNA）是一类小的非编码RNA，它们在转录后调节基因表达，并因其在疾病进展调节和疾病活动生物标志物方面的作用而被认可，这些疾病包括神经疾病、癌症和心血管疾病（CVD）。通常，使用定量实时PCR（qRT-PCR）来评估miRNA丰度，然而，用于miRNA的qRT-PCR可能劳动强度大、耗时，并且在检测成熟与前体形式的miRNA时可能缺乏特异性。在这里，我们描述了一种用于miRNA评估的新型双分子信标方法，该方法可以在一次测定中区分和定量成熟与前体形式的miRNA，这是将miRNA用作疾病生物标志物的一个基本特征。使用这种方法，我们发现具有DNA或组合锁核酸（LNA）-DNA骨架的分子信标可以检测低丰度（<1 nM）的成熟和前体miRNA（pre-miRNA）。双分子信标测定法在评估含有成熟和前体miRNA混合群体的样品中的miRNA丰度时是准确的。相比之下，qRT-PCR和单分子信标测定法高估了miRNA丰度。此外，双分子信标测定法比传统qRT-PCR劳动强度小，特异性提高了10-25%。我们的数据表明，基于双分子信标的方法比以前的方法更精确、更特异，并且有望成为评估生物样品中miRNA水平的标准方法。
