A new method for multiplex detection of micro-RNAs (miR-ID)

描述（申请人提供）：一种用于多重检测微小RNA（miR-ID）的新方法。ABSRACT微小RNA（miRNAs）和其他短的非编码RNA发挥不同的生物学功能，并且独特的miRNA表达模式与各种类型的癌症和病毒感染相关。因此，miRNAs可能被认为是潜在的诊断生物标志物以及潜在的药物靶点。近年来，人们发展了多种检测miRNAs及其表达谱的方法。然而，这些方法中没有一种能够以相同的准确度和灵敏度定量不同的miRNA，并且都存在诸如费力的处理和纯化步骤以及昂贵的设备和试剂的问题。在这里，我们提出了一种新的方法，我们称之为miR-ID，它允许通过信号（而不是目标）扩增来快速，准确，灵敏和高度多重量化短靶RNA，如miRNA。miR-ID方法也可能比其他可用方法更简单和昂贵。MiR-ID采用含有miRNA特异性捕获序列以及信号序列的探针。预期该探针直接从核酸提取物或细胞裂解物中有效地和序列特异性地捕获靶miRNA。然后以可以通过qPCR定量的方式处理探针-miRNA复合物。所提出的检测方法不需要对miRNA进行化学或酶标记，避免了可能由miRNA连接和逆转录引入的序列依赖性偏倚，与来自各种供应商的qPCR机器兼容，并且允许大规模多路复用。在第一阶段，我们将测试几种不同的捕获探针设计，并寻求灵敏度，序列特异性，低成本和工作流程时间的最佳组合。我们还将通过定量细胞miRNA并将我们的结果与用其他方法获得的已发表数据进行比较来验证该方法在“真实世界”环境中的灵敏度和特异性。在第二阶段，我们计划开发一种用于前列腺癌检测以及监测疾病进展和治疗反应的诊断方法。公共卫生相关性：前列腺癌是美国男性癌症死亡的第二大原因。该应用的目标是基于构成有希望的生物标志物的一组microRNA的定量来早期诊断这种疾病。micro-RNA检测方法具有新的特点，比目前可用的其他micro-RNA检测方法具有更好的灵敏度和选择性。

DESCRIPTION (provided by applicant): A new method for multiplex detection of micro-RNAs (miR-ID) ABSRACT MicroRNAs (miRNAs) and other short non-coding RNAs play diverse biological functions, and distinctive miRNA expression patterns are associated with various types of cancer and viral infections. Thus, miRNAs may be considered as potential diagnostic biomarkers as well as potential drug targets. Many different methods for detecting miRNAs and profiling their expression patterns have been developed recently. However, none of these methods is capable of quantifying different miRNAs with equal accuracy and sensitivity, and all suffer from problems such as laborious handling and purification steps, and expensive equipment and reagents. Here we propose a novel method, which we call miR-ID, that allows fast, accurate, sensitive, and highly multiplexable quantification of short target RNAs such as miRNAs through signal (rather than target) amplification. The miR-ID approach is also potentially less complex and expensive than alternative available methods. MiR-ID employs a probe that contains a miRNA-specific capture sequence as well as signal sequences. This probe is expected to efficiently and sequence-specifically capture target miRNAs directly from nucleic acid extracts or cell lysates. The probe-miRNA complex is then processed in such a way that it can be quantified by qPCR. The proposed detection method has no need for chemical or enzymatic labeling of miRNAs, avoids sequence-dependent bias that could be introduced by miRNA ligation and reverse transcription, is compatible with qPCR machines from a variety of vendors, and allows massive multiplexing. In Phase I, we will test several different designs of capture probes and seek the best combination of sensitivity, sequence-specificity, low cost, and workflow time. We will also verify the sensitivity and specificity of the method in a "real-world" setting by quantifying cellular miRNAs and comparing our results with published data obtained with other methods. In Phase II, we plan to develop a diagnostic assay for prostate cancer detection as well as for monitoring progression of the disease and response to therapy. PUBLIC HEALTH RELEVANCE: Prostate cancer is the second leading cause of cancer deaths among American men. The goal of this application is early diagnosis of this disease based on quantification of a panel of microRNAs that constitutes a promising biomarker. The method of micro-RNA detection has novel features that promise better sensitivity and selectivity than other micro-RNA assays currently available.