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

摘要MicroRNAs （miRNAs）和其他短链非编码rna具有多种生物学功能，其独特的miRNA表达模式与多种类型的癌症和病毒感染有关。因此，mirna可以被认为是潜在的诊断性生物标志物以及潜在的药物靶点。最近开发了许多不同的检测mirna和分析其表达模式的方法。然而，这些方法都不能以相同的精度和灵敏度定量不同的mirna，而且都存在处理和纯化步骤费力、设备和试剂昂贵等问题。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（而不是靶标）扩增对短靶rna（如miRNAs）进行快速、准确、敏感和高度可复用的量化。miR-ID方法也可能比其他可用方法更不复杂和昂贵。MiR-ID采用包含mirna特异性捕获序列和信号序列的探针。该探针有望有效和序列特异性捕获目标mirna直接从核酸提取物或细胞裂解物。然后将探针- mirna复合物以qPCR定量的方式进行处理。所提出的检测方法不需要对miRNA进行化学或酶标记，避免了miRNA连接和逆转录可能引入的序列依赖性偏差，与来自各种供应商的qPCR机器兼容，并允许大量复用。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并将我们的结果与其他方法获得的已发表数据进行比较，在“现实世界”环境中验证该方法的敏感性和特异性。在II期，我们计划开发一种前列腺癌检测的诊断方法，以及监测疾病的进展和对治疗的反应。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。这一应用的目的是基于一组microrna的量化来早期诊断这种疾病，这些microrna构成了一种有前途的生物标志物。微rna检测方法具有新的特点，比目前可用的其他微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.