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

本提案旨在开发一种基于人类血清中发现的microRNA （miRNA）表达模式的癌症诊断方法。越来越多的证据表明，miRNA的表达模式和这些信号在循环的细胞外区室中的出现是肿瘤细胞增殖的标志。我们的初步结果已经显示出区分5种不同类型癌症患者血清的清晰能力。在涉及246例患者衍生血清样本的数据集中，我们能够以76%的灵敏度和84%的特异性区分癌症衍生血清和正常对照。这些数据虽然很有希望，但也向我们表明，为了解决这些不同的miRNA表达模式，需要更敏感的分析。为此，我们建议开发一种多路挂锁探针检测，与微阵列检测相结合，可以同时检测大多数已知的人类mirna。我们独特的定制DNA微阵列合成平台使我们既可以生产挂锁探针，也可以从放大的混合物中检测信号。最后，我们将利用该实验研究保护增殖肿瘤细胞分泌的miRNA分子的细胞外隔室或结构。在II期研究中，我们将应用该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）的表达模式，重复我们之前的工作。

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.