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

描述(申请人提供)：一种新的多重检测microRNAs(miR-ID)的方法ABSRACT microRNAs(MiRNAs)和其他短的非编码RNA发挥着不同的生物学功能，独特的miRNA表达模式与各种类型的癌症和病毒感染有关。因此，miRNAs可以被认为是潜在的诊断生物标志物和潜在的药物靶点。最近开发了许多不同的方法来检测miRNAs并分析其表达模式。然而，这些方法都不能以同样的准确度和灵敏度定量不同的miRNAs，并且都存在着操作和纯化步骤繁琐以及昂贵的设备和试剂等问题。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、灵敏和高度可复用地量化短目标RNA，例如miRNAs。与其他可用的方法相比，miR-ID方法也可能不那么复杂和昂贵。MIR-ID使用包含miRNA特定捕获序列和信号序列的探针。该探针有望直接从核酸提取物或细胞裂解物中高效地、序列特异性地捕获目标miRNAs。然后，对探针-miRNA复合体进行处理，使其可以通过qPCR进行定量。所提出的检测方法不需要对miRNAs进行化学或酶标记，避免了miRNA连接和反转录可能导致的序列依赖偏差，与各种供应商的qPCR机兼容，并允许大规模多路复用。在第一阶段，我们将测试几种不同设计的捕获探针，并寻求灵敏度、序列特异性、低成本和工作时间的最佳组合。我们还将通过对细胞miRNAs进行量化，并将我们的结果与用其他方法获得的公开数据进行比较，来验证该方法在“真实世界”环境中的敏感性和特异性。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。这项应用的目标是基于对一组构成有前景的生物标记物的microRNAs的量化，对这种疾病进行早期诊断。与现有的其他微量RNA检测方法相比，该方法具有新的特点，有望获得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.