PCR-free Multiplexed Detection of Circulating miRNA in Blood

血液中循环 miRNA 的无 PCR 多重检测

• 批准号: 8432948
• 负责人: Tza-Huei Jeff Wang
• 金额: $ 23.22万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-17 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8432948
• 关键词: Address; Apoptosis; Benchmarking; Biological; Biological Markers; Blood; Blood Circulation; Blood specimen; Body Fluids; Cancer Detection; Cancer Prognosis; Cell physiology; Cells; Clinical; Clinical assessments; Code; DNA; DNA Ligation; Detection; Diagnostic; Early Diagnosis; Esophageal Adenocarcinoma; Functional RNA; Gel; Gene Expression; Generations; Genes; Goals; Gold; Length; Ligation; Lighting; Liquid substance; Malignant Neoplasms; Malignant neoplasm of esophagus; Messenger RNA; Methods; MicroRNAs; Microfluidic Microchips; Microfluidics; Modality; Mole the mammal; Molecular; Molecular Profiling; Neoplasm Metastasis; Neoplasms; Nucleotides; Oligonucleotide Probes; Patients; Performance; Physiological; Plasma; Play; Process; Publications; RNA; RNA Degradation; Reaction; Regulator Genes; Resolution; Sample Size; Sampling; Scheme; Sensitivity and Specificity; Serum; Specificity; Spectrum Analysis; Stream; Techniques; Technology; Testing; Tissues; Validation; base; cancer diagnosis; meetings; prognostic; single molecule; submicron; tumor; tumorigenesis

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. PUBLIC HEALTH RELEVANCE: The goal of the proposed project is to develop a single-molecule length-coding platform capable of highly sensitive and multiplexed analysis of panels of miRNAs in the blood stream, addressing the unmet clinical need of using circulating miRNAs as cancer biomarkers.

描述（由申请人提供）：微小RNA（miRNA）是短核苷酸（约20 nt），在几乎所有细胞过程（包括分化、增殖和凋亡）中充当基因表达的调节因子。在肿瘤中，miRNA 已被证明在癌症转移和肿瘤发生等过程中发挥关键作用。由于单个 miRNA 可以调节许多 mRNA，因此一个 miRNA 的失调可能会产生深远的生物学后果，并且一小部分 miRNA 可能足以用于诊断目的。最近发现血流中存在循环 miRNA，进一步提高了 miRNA 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 miRNA 体积小且受到外泌体外壳的保护，它们可以强有力地抵抗组织和血液中的 RNA 降解。这些特征使 miRNA 成为癌症诊断和预后的令人兴奋的目标。由于无细胞、循环 miRNA 的生理浓度非常低，目前检测这些靶标的方法主要依赖于高灵敏度的 RT-qPCR。然而，RT-qPCR 通常仅限于单重分析，而 miRNA 的临床评估需要以快速且廉价的方式对一组（例如 10-100）miRNA 进行定量。对每个样本进行大量单独的 PCR 成本高昂，并且需要大量的 miRNA，而这很难从血液样本中获得。另一方面，现有的多重技术（例如 miRNA 微阵列）严重缺乏检测体液中这些循环 miRNA 组所需的灵敏度。在这个项目中，我们建议开发一个单分子长度编码平台，以满足对循环 miRNA 的高灵敏度和多重检测的未满足的临床需求。该平台采用基于连接的分子长度编码方案来生成 miRNA 特异性长度编码链，通过大小分离来破译这些链，以促进多重检测。它利用柱面照明共焦光谱通过单分子计数对低浓度目标进行定量，实现高灵敏度和定量精度。此外，还将开发一种微流体装置，将多个微升大小的样品同时浓缩到皮升大小的塞子中，在亚微米通道中进行阵列分离，以提高分离分辨率和分析通量。最后，我们将通过确定分析灵敏度来验证所提出的平台 以及使用掺有合成 miRNA 序列的对照血清样品的特异性。我们的目标是实现 PCR 等效灵敏度 <10-22 摩尔，无关 miRNA 的特异性 >1000:1，相关 miRNA 的特异性 >100:1。将通过在单次反应中使用 200 mL 血清分析晚期食管腺癌患者（20 个样本）和健康对照（20 个样本）血清中的一组 20 个 miRNA 来进行临床样本验证。结果将与使用 4 mL 血清分成 20 个独立单重反应的 RT-qPCR 获得的结果进行比较。 公共健康相关性：拟议项目的目标是开发一个单分子长度编码平台，能够对血流中的 miRNA 组进行高灵敏度和多重分析，解决使用循环 miRNA 作为癌症生物标志物的未满足的临床需求。