ADAPTING PADLOCK-PROBE DETECTION SYSTEMS TO NCRNA DETECTION FOR CANCER DIAGNOSTI

该提案旨在开发基于人血清中发现的microRNA（miRNA）表达模式的癌症诊断。越来越多的证据表明，miRNA表达的模式和这些信号在循环细胞外区室中的出现是肿瘤细胞增殖的标志。我们的初步结果显示了区分5种不同类型癌症的癌症患者血清的明确能力。在涉及246个患者来源的血清样品的数据集中，我们能够区分癌症来源的血清与正常对照，灵敏度为76%，特异性为84%。这些数据虽然有希望，但也向我们表明，为了解决这些不同的miRNA表达模式，需要更灵敏的检测方法。为此，我们建议开发一种多重挂锁探针检测，与微阵列检测集成，同时检测大多数已知的人类miRNA。我们独特的可定制DNA微阵列合成平台使我们能够生产挂锁探针并检测来自扩增混合物的信号。最后，我们将使用这种检测方法，研究细胞外区室或结构，保护增殖肿瘤细胞分泌的miRNA分子。在第二阶段，我们将应用这种检测方法从正常和癌症患者血清样本中寻找miRNA和其他非编码RNA（ncRNA）表达的模式，重复我们以前的工作。

This proposal seeks to develop a cancer diagnostic based on microRNA (miRNA) expression patterns found in human serum. Mounting evidence is showing that patterns of miRNA expression and the appearance of these signals in circulating extra-cellular compartments are hallmarks of proliferating tumor cells. Our preliminary results have shown a clear ability to distinguish sera derived from cancer patients for 5 different types of cancer. In a data set involving 246 patient-derived serum samples, we were able to distinguish cancer derived sera from normal controls with a sensitivity of 76% and a specificity of 84%. These data, though promising, also indicated to us that a more sensitive assay is necessary in order to resolve these distinct patterns of miRNA expression. To this end, we propose the development of a multiplexed padlock probe assay, integrated with microarray detection that will simultaneously detect most of the known human miRNAs. Our unique customizable DNA microarray synthesis platform allows us to both produce the padlock probes and to detect signal from the amplified mixture. Finally, using this assay, we will investigate the extracellular compartment or structure that protects the miRNA molecules that are secreted by proliferating tumor cells. In phase II, we will apply this assay to look for patterns of miRNA and other non-coding RNA (ncRNA) expression from normal and cancer patient serum samples, repeating our previous work with this novel assay.