Department of Chemistry, University of California, Riverside, California 92521, USA.
Anal Chem. 2009 Dec 1;81(23):9723-9. doi: 10.1021/ac901983s.
Small RNA molecules are effective regulators of gene expression, and the expression signature of one subgroup of small RNA, the microRNA (miRNA), has been linked to disease development and progression. Therefore, detection of small RNA in biological samples will greatly improve the understanding of their functions and render effective tools to researchers for cellular process control and disease prevention. To solve the challenges in detecting the low-abundance and short strand-length of small RNA molecules, we designed a ligation-assisted binding assay and applied the cation exchange-based fluorescence amplification (CXFluoAmp) method developed in our group for detection. Nonfluorescent, ionic nanocrystals (NCs) of CdSe were conjugated to detection probes and immobilized onto the array surface via ligation with the target small RNA, miR21, which bound to the capture probe complimentarily. Each binding event induced by one target miR21 molecule was then amplified by the release of thousands of Cd2+ from one NC. The free Cd2+ immediately turned on the fluorescence of thousands of fluorogenic Rhod-5N molecules. With such a powerful signal amplification strategy, our assay achieved a limit of detection (LOD) of 35 fM and signals were detectable with analyte concentrations spanning over 7 orders of magnitude. We also identified the differential expression of miR21 in total RNA extracts from healthy breast tissue and diseased cells. Furthermore, our detection scheme demonstrated good specificity in small RNA detection, because significant signal intensity could be observed from small RNAs with one or two nucleotides difference in sequences. Thus, our assay has great application potential for disease diagnosis relying on miRNA biomarkers, or in small RNA expression profiling for new target discovery and functional study.
小分子 RNA 是基因表达的有效调控因子，其中一组小分子 RNA，即 microRNA（miRNA）的表达特征与疾病的发生和发展有关。因此，在生物样本中检测小分子 RNA 将极大地提高对其功能的理解，并为研究人员提供有效的工具来控制细胞过程和预防疾病。为了解决检测小分子 RNA 低丰度和短链长的挑战，我们设计了一种连接辅助结合测定法，并应用我们小组开发的基于阳离子交换的荧光扩增（CXFluoAmp）方法进行检测。非荧光、离子纳米晶体（NCs）的 CdSe 与检测探针缀合，并通过与互补的捕获探针结合的靶小分子 RNA，miR21 进行连接而固定在阵列表面上。每个由一个靶 miR21 分子引起的结合事件随后通过从一个 NC 释放数千个 Cd2+而被放大。游离的 Cd2+立即开启数千个荧光 Rhod-5N 分子的荧光。通过这种强大的信号放大策略，我们的测定法实现了 35 fM 的检测限（LOD），并且可以检测到分析物浓度跨越 7 个数量级的信号。我们还鉴定了来自健康乳腺组织和患病细胞的总 RNA 提取物中 miR21 的差异表达。此外，我们的检测方案在小 RNA 检测中表现出良好的特异性，因为在序列上有一个或两个核苷酸差异的小 RNA 上可以观察到显著的信号强度。因此，我们的测定法在基于 miRNA 生物标志物的疾病诊断或用于新靶标发现和功能研究的小 RNA 表达谱分析方面具有巨大的应用潜力。
