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Total internal reflection-based single-vesicle in situ quantitative and stoichiometric analysis of tumor-derived exosomal microRNAs for diagnosis and treatment monitoring.
Theranostics ( IF 12.4 ) Pub Date : 2019-01-01 , DOI: 10.7150/thno.33683 Dinggeng He 1, 2 , Huizhen Wang 2 , See-Lok Ho 1 , Hei-Nga Chan 1 , Luo Hai 2 , Xiaoxiao He 2 , Kemin Wang 2 , Hung-Wing Li 1, 2
Theranostics ( IF 12.4 ) Pub Date : 2019-01-01 , DOI: 10.7150/thno.33683 Dinggeng He 1, 2 , Huizhen Wang 2 , See-Lok Ho 1 , Hei-Nga Chan 1 , Luo Hai 2 , Xiaoxiao He 2 , Kemin Wang 2 , Hung-Wing Li 1, 2
Affiliation
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Purpose: Exosomes (EXs) have been increasingly recognized as natural nanoscale vehicles for microRNA (miRNA)-based cell-cell communication and an ideal source of miRNA biomarkers in bodily fluids. Current methods allow bulk analysis of the miRNA contents of EXs, but these approaches are not suitable for the in situ stoichiometry of exosomal miRNAs and fail to reveal phenotypic heterogeneity at the single-vesicle level. This study aimed to develop a single vesicle-based, mild, precise, but versatile method for the in situ quantitative and stoichiometric analysis of exosomal miRNAs. Methods: A total internal reflection fluorescence (TIRF)-based single-vesicle imaging assay was developed for direct visualization and quantification of the single-vesicles of EXs and their miRNA contents in serum microsamples. The assay uses co-delivery of inactive split DNAzymes and fluorescence-quenched substrates into nanosized EXs treated with streptolysin O to produce a target miRNA-activated catalytic cleavage reaction that amplifies the readout of fluorescence signal. We perform the in situ quantitative and stoichiometric analysis of serum exosomal hsa-miRNA-21 (miR-21), a common cancer biomarker, by using the developed TIRF imaging assay. Results: The TIRF imaging assay for serum exosomal miR-21 can distinguish cancer patients from healthy subjects with better performance than conventional real-time polymerase chain reaction (PCR) assay. The exosomal miR-21 level in serum is also informative for monitoring tumor progression and responses to treatment. Moreover, the TIRF assays can readily determine the precise stoichiometry of target exosomal miRNA contents in situ by delivering molecular beacon (MB) probes into EXs. Conclusions: The created TIRF imaging platform shows high applicability to serve as a universal and useful tool for the single-vesicle in situ quantitative and stoichiometric analysis of other disease-associated exosomal miRNAs markers and provide valuable insight into the physiological relevance of EX-mediated miRNA communication.
中文翻译：
基于全内反射的肿瘤来源的外泌体microRNA的单囊泡原位定量和化学计量分析，用于诊断和治疗监测。
目的：外来体（EXs）已被越来越多地视为基于microRNA（miRNA）的细胞间通信的天然纳米载体，并且是体液中miRNA生物标记物的理想来源。当前的方法允许大量分析EX的miRNA含量，但这些方法不适用于外泌体miRNA的原位化学计量，并且无法揭示单囊泡水平的表型异质性。这项研究旨在开发一种基于小泡的，温和，精确但通用的方法，用于外泌体miRNA的原位定量和化学计量分析。方法：建立了基于全内反射荧光（TIRF）的单囊泡成像测定法，用于直接可视化和定量血清微量样品中EX的单囊泡及其miRNA含量。该测定法将无活性的分裂DNA酶和荧光淬灭的底物共同输送到经链球菌溶血素O处理的纳米EX中，以产生目标miRNA激活的催化裂解反应，从而放大了荧光信号的读数。通过使用开发的TIRF成像分析，我们对血清外泌体hsa-miRNA-21（miR-21）（一种常见的癌症生物标记物）进行原位定量和化学计量分析。结果：血清外泌体miR-21的TIRF成像测定法可以比常规实时聚合酶链反应（PCR）测定法更好地将癌症患者与健康受试者区分开。血清中的外泌体miR-21水平也有助于监测肿瘤的进展和对治疗的反应。而且，通过将分子信标（MB）探针传递到EX中，TIRF分析可以轻松地就地确定目标外泌体miRNA含量的精确化学计量。结论：所创建的TIRF成像平台具有很高的适用性，可作为其他疾病相关外泌体miRNA标记的单囊原位定量和化学计量分析的通用工具，并为了解EX介导的miRNA的生理相关性提供有价值的见解。沟通。
更新日期：2019-01-01
中文翻译：
基于全内反射的肿瘤来源的外泌体microRNA的单囊泡原位定量和化学计量分析，用于诊断和治疗监测。
目的：外来体（EXs）已被越来越多地视为基于microRNA（miRNA）的细胞间通信的天然纳米载体，并且是体液中miRNA生物标记物的理想来源。当前的方法允许大量分析EX的miRNA含量，但这些方法不适用于外泌体miRNA的原位化学计量，并且无法揭示单囊泡水平的表型异质性。这项研究旨在开发一种基于小泡的，温和，精确但通用的方法，用于外泌体miRNA的原位定量和化学计量分析。方法：建立了基于全内反射荧光（TIRF）的单囊泡成像测定法，用于直接可视化和定量血清微量样品中EX的单囊泡及其miRNA含量。该测定法将无活性的分裂DNA酶和荧光淬灭的底物共同输送到经链球菌溶血素O处理的纳米EX中，以产生目标miRNA激活的催化裂解反应，从而放大了荧光信号的读数。通过使用开发的TIRF成像分析，我们对血清外泌体hsa-miRNA-21（miR-21）（一种常见的癌症生物标记物）进行原位定量和化学计量分析。结果：血清外泌体miR-21的TIRF成像测定法可以比常规实时聚合酶链反应（PCR）测定法更好地将癌症患者与健康受试者区分开。血清中的外泌体miR-21水平也有助于监测肿瘤的进展和对治疗的反应。而且，通过将分子信标（MB）探针传递到EX中，TIRF分析可以轻松地就地确定目标外泌体miRNA含量的精确化学计量。结论：所创建的TIRF成像平台具有很高的适用性，可作为其他疾病相关外泌体miRNA标记的单囊原位定量和化学计量分析的通用工具，并为了解EX介导的miRNA的生理相关性提供有价值的见解。沟通。
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