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: 8546323
• 负责人: Tza-Huei Jeff Wang
• 金额: $ 21.83万
• 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/8546323
• 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.

描述（由申请人提供）：微小RNA（miRNAs）是短核苷酸（~ 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剂。在肿瘤中，miRNA已被证明在癌症过程如转移和肿瘤发生中起关键作用。由于单个miRNA可以调节许多mRNA，因此一个miRNA的失调可能具有深远的生物学后果，并且一小组miRNA可能足以用于诊断目的。最近发现血液中存在循环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，由于其尺寸小且受到外泌体壳的保护，miRNA可以强烈抵抗组织和血液中的RNA降解。这些特征使得miRNA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miRNA以非常低的生理浓度存在，目前检测这些靶标的方法主要依赖于高度灵敏的RT-qPCR。然而，RT-qPCR通常限于单重分析，而miRNA的临床评估需要以快速和廉价的方式定量一组（例如10 -100）miRNA。对每个样本使用大量单独的PCR是昂贵的，并且需要大量的miRNA，而这很难从血液样本中获得。另一方面，现有的多路复用技术如miRNA微阵列在检测体液中这些循环miRNA组的必要灵敏度方面严重缺乏。在这个项目中，我们建议开发一个单分子长度的编码平台，以解决未满足的临床需要，高灵敏度和多重检测循环miRNA。该平台采用基于连接的分子长度编码方案来产生miRNA特异性长度编码链，其通过大小分离来解密以促进多重检测。它利用柱面照明共聚焦光谱通过单分子计数来定量低浓度目标，实现高灵敏度和定量准确性。此外，将开发一种微流控装置，以同时将多个微升大小的样品浓缩到皮升大小的塞中，用于在亚微米通道中进行阵列分离，以提高分离的分辨率和分析的通量。最后，我们将通过确定分析灵敏度来验证所提出的平台 和特异性，使用掺入合成miRNA序列的对照血清样品。我们的目标是实现<10-22摩尔的PCR等效灵敏度，对于无关miRNA的特异性>1000：1，对于相关miRNA的特异性> 100：1。将通过在单个反应中使用d 200 mL血清分析晚期食管腺癌患者（20个样品）和健康对照（20个样品）血清中的一组20种miRNA来进行临床样品的验证。将结果与RT-qPCR获得的结果进行比较，RT-qPCR使用4 mL血清分为20个单独的单重反应。