PCR-free Multiplexed Detection of Circulating miRNA in Blood

描述(申请人提供)：microRNAs(MiRNAs)是一种短核苷酸(~20nt)，在几乎所有的细胞过程中发挥基因表达的调节作用，包括分化、增殖和凋亡。在肿瘤中，miRNAs已被证明在肿瘤转移和肿瘤发生等癌症过程中发挥关键作用。由于单个miRNA可以调节许多mRNAs，因此一个miRNA的失调可能会产生深远的生物学后果，而且一小组miRNAs可能足以用于诊断目的。最近发现在血流中存在循环中的miRNAs，这进一步提高了miRNAs作为远程癌症检测的非侵入性生物标志物的潜力。此外，由于miRNAs体积小，并且在外体外壳中具有保护作用，因此可以很好地抵抗组织和血液中的RNA降解。这些特性使miRNAs成为癌症诊断和预后的令人兴奋的靶点。由于无细胞的、循环中的miRNAs存在于非常低的生理浓度下，目前检测这些靶标的方法主要依赖于高灵敏的RT-qPCR。然而，RT-qPCR通常仅限于单链分析，而对miRNAs的临床评估要求以快速和廉价的方式对一组(例如10-100)miRNAs进行定量。为每个样本使用大量单独的PCR是昂贵的，并且需要大量的miRNA，这是很难从血液样本中获得的。另一方面，现有的多路技术，如miRNA微阵列，严重缺乏检测体液中这些循环中的miRNA面板所需的灵敏度。在这个项目中，我们建议开发一个单分子长度编码平台，以满足临床对循环miRNA的高灵敏度和多重检测的需求。该平台采用基于连接的分子长度编码方案来生成miRNA特定长度编码的链，这些链通过大小分离进行解密，以便于多路复用检测。它利用柱面照明共聚焦光谱，通过单分子计数对低浓度目标进行定量，实现了高灵敏度和定量精度。此外，还将开发一种微流控装置，将多个微升大小的样品同时浓缩到皮升大小的塞子中，以便在亚微米通道中进行阵列分离，以提高分离分辨率和分析吞吐量。最后，我们将通过确定分析灵敏度来验证所提出的平台 以及使用添加了合成miRNA序列的对照血清样本的特异性。我们的目标是达到&lt；10-22摩尔的PCR等效灵敏度，对无关miRNA的&gt；1000：1的特异性和对相关miRNA的&gt；100：1的特异性。临床样本的验证将通过在一次反应中使用200毫升血清分析晚期食管腺癌患者(20例)和健康对照组(20例)血清中的20个miRNAs来完成。这一结果将与RT-qPCR的结果进行比较，该方法使用4毫升血清，分成20个单独的单链反应。

DESCRIPTION (provided by applicant): Micro RNAs (miRNAs) are short nucleotides (~ 20 nt) that act as regulators of gene expression in nearly all cellular processes including differentiatio, proliferation, and apoptosis. In tumors, miRNAs have been shown to play key roles in cancer processes such as metastasis and tumorigenesis. Since a single miRNA can regulate many mRNAs, dysregulation of one miRNA can have far-reaching biological consequences and that a small panel of miRNAs may suffice for diagnostic purposes. Recent discovery of the existence of circulating miRNAs in the blood stream further raises the potential of miRNAs as noninvasive biomarkers for remote cancer detection. In addition, due to their small size and protection within an exosomal shell, miRNAs robustly resist RNA degradation in tissue and blood. These features make miRNAs exciting targets for cancer diagnosis and prognosis. Since cell-free, circulating miRNAs exist at very low physiological concentrations, current methods to detect these targets predominantly rely on highly sensitive RT-qPCR. However, RT-qPCR is generally limited to single-plex analyses while clinical assessment of miRNAs requires that a panel (e.g.10-100) miRNAs to be quantified in a rapid and inexpensive manner. Employing a large number of separate PCRs for each sample is costly and requires large amounts of miRNA, which is difficult to obtain from blood samples. On the other hand, existing multiplexed technologies such as miRNA microarrays are woefully lacking in the requisite sensitivity to detect these circulating miRNA panels in body fluids. In this project, we propose to develop a single molecule length coding platform to address the unmet clinical need for highly sensitive and multiplexed detection of circulating miRNA. The platform employs a ligation- based molecular length coding scheme to generate miRNA-specific length-encoded strands that are deciphered by size separation to facilitate multiplexed detection. It utilizes cylindrical illumination confocal spectroscopy to quantify low concentration targets through single molecule counting, achieving high sensitivity and quantitative accuracy. In addition, a microfluidic device will be developed to simultaneously concentrate multiple microliter-sized samples into picoliter-sized plugs for arrayed separation in sub-micron channels to enhance both the resolution of separation and the throughput of analysis. Finally, we will validate the proposed platform by determining the analytical sensitivity and specificity using control serum samples spiked with synthetic miRNA sequences. We aim to achieve PCR-equivalent sensitivity of <10-22 mole, specificity of >1000:1 for unrelated miRNA and > 100:1 for related miRNA. Validation with clinical samples will be performed by analyzing a panel of 20 miRNAs in the serum of patients with advanced esophageal adenocarcinoma (20 samples) and healthy controls (20 samples) using d 200 mL of serum in a single reaction. The result will be compared to that obtained by RT-qPCR using 4 mL of serum split into 20 separate single-plex reactions.