Neely Lori A, Patel Sonal, Garver Joanne, Gallo Michael, Hackett Maria, McLaughlin Stephen, Nadel Mark, Harris John, Gullans Steve, Rooke Jenny
US Genomics, 12 Gill Street, Suite 4700, Woburn, Massachusetts 01801, USA.
Nat Methods. 2006 Jan;3(1):41-6. doi: 10.1038/nmeth825.
MicroRNAs (miRNA) are short endogenous noncoding RNA molecules that regulate fundamental cellular processes such as cell differentiation, cell proliferation and apoptosis through modulation of gene expression. Critical to understanding the role of miRNAs in this regulation is a method to rapidly and accurately quantitate miRNA gene expression. Existing methods lack sensitivity, specificity and typically require upfront enrichment, ligation and/or amplification steps. The Direct miRNA assay hybridizes two spectrally distinguishable fluorescent locked nucleic acid (LNA)-DNA oligonucleotide probes to the miRNA of interest, and then tagged molecules are directly counted on a single-molecule detection instrument. In this study, we show the assay is sensitive to femtomolar concentrations of miRNA (500 fM), has a three-log linear dynamic range and is capable of distinguishing among miRNA family members. Using this technology, we quantified expression of 45 human miRNAs within 16 different tissues, yielding a quantitative differential expression profile that correlates and expands upon published results.
微小RNA（miRNA）是短的内源性非编码RNA分子，其通过调节基因表达来调控细胞分化、细胞增殖和凋亡等基本细胞过程。理解miRNA在这种调控中作用的关键是一种能够快速、准确地定量miRNA基因表达的方法。现有方法缺乏灵敏度、特异性，并且通常需要前期富集、连接和/或扩增步骤。直接miRNA检测法将两种光谱可区分的荧光锁定核酸（LNA）-DNA寡核苷酸探针与感兴趣的miRNA杂交，然后在单分子检测仪器上直接计数标记分子。在本研究中，我们表明该检测法对飞摩尔浓度的miRNA（500 fM）敏感，具有三个对数的线性动态范围，并且能够区分miRNA家族成员。使用该技术，我们定量了16种不同组织中45种人类miRNA的表达，产生了一个定量差异表达谱，该谱与已发表的结果相关并扩展了其内容。
