PCR-free UPLC-MS/MS based quantitative assay of microRNAs

基于无 PCR UPLC-MS/MS 的 microRNA 定量分析

• 批准号: 10403806
• 负责人: YIMING LIU
• 金额: $ 13.72万
• 依托单位: JACKSON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-15 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10403806
• 关键词: 1-Methyl-4-phenylpyridinium; Acids; Adenine; Affinity; Azacitidine; Biological; Biological Assay; Biological Markers; Biology; Biomedical Research; Breast Cancer Cell; Breast Epithelial Cells; Calibration; Cell Culture Techniques; Cell Line; Cell model; Cells; Characteristics; Chemistry; Cisplatin; Clinical; DU145; Data; Development; Disadvantaged; Discrimination; Disease; Enzymes; Evaluation; Exclusion; Fluorescence; Fostering; Future; Goals; Hydrolysis; Intoxication; Ion Exchange; Label; Light; Liquid Chromatography; MCF10A cells; MCF7 cell; MDA MB 231; Magnetism; Measurement; Medical; Methodology; Methods; MicroRNAs; Modeling; Nature; Nerve Degeneration; Neuroblastoma; Neurons; PC12 Cells; PC3 cell line; Parkinson Disease; Pathologic; Performance; Pharmacotherapy; Phase; Physiological; Polyadenylation; Polymerase Chain Reaction; Polynucleotide Adenylyltransferase; Preparation; Principal Investigator; Procedures; Process; Prostate; RNA; Recovery; Reporting; Research; Research Activity; Reverse Transcription; Sampling; Serum; Signal Transduction; Solid; Specimen; Students; Surface; Techniques; Testing; Uncertainty; Universities; Urine; Validation; Work; analytical method; base; cancer biomarkers; cancer cell; cost; cost effective; cost effectiveness; design; disease diagnosis; exosome; improved; innovation; logarithm; magnetic beads; microRNA biomarkers; novel; programs; skills; tandem mass spectrometry; two-dimensional

Project Summary Aberrant expression of microRNAs has been found associated with pathological conditions. Over the past years many putative miRNA-based disease biomarkers have been reported, but none of them has been fully validated (e.g., for FDA approval). This is largely because of the lack of an analytical methodology that offers accurate, repeatable, and cost-effective quantification of microRNAs present at very low levels in biological specimen. The current golden standard for microRNA assay (i.e., quantitative reverse transcription polymerase chain reaction, RT-qPCR) offers “a relative quantification” and is very high in assay cost (>$15 of consumables /per assay). After all, all PCR-based quantitative assays deploy a calibration curve established between fluorescence signal and the logarithm of microRNA concentration (instead of microRNA concentration), which by nature produces less accurate quantitation results and exponentially amplifies the uncertainties contained in fluorescence signal measurements. The goal of the research is to eliminate current limitations in quantitative assay of microRNAs, thus fostering the biomedical research and full validation of these emerging disease biomarkers. We propose herein a novel analytical strategy for “absolute quantification” of target microRNAs based on robust and popular ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in combination with affinity magnetic solid phase extraction and isothermal signal amplification. The strategy involves the following PCR- free workflow: 1) target microRNA is extracted /enriched from a biological sample by using an ssDNA probe- magnetic bead conjugate; 2) solid surface polyadenylation of target microRNA captured by poly(A) polymerase with 13C-labeled adenine; and 3) UPLC-MS/MS determination of 13C-labeled adenine after acid hydrolysis of the extended target microRNA. The quantitative assay is expected to have the following assay characteristics: high sensitivity (LOQs < 1pM, a physiologically relevant level), high accuracy (recovery ≥ 95%), good repeatability (RSD ≤ 5%), the capability of single base mismatch discrimination, no need for a total RNA isolation in the assay, and cost-effectiveness (a total cost of consumables < $1.5 per assay. Implementation of this analytical method will have a profound impact on microRNA biomedical research. The project proposed fits the research concentration at Jackson State University and will be able to draw students from the Chemistry and Biology programs. The participating students will acquire lab skills including cell culture, biological sample preparation, instrumental analysis, and scientific data process /presentation through research activities. In general, the project will help to develop and to sustain research excellence at JSU (an HBCU), and thus contribute to the diversity of our future research workforce.

项目概要 在过去的几年里，人们发现 microRNA 的异常表达与病理状况有关。 许多假定的基于 miRNA 的疾病生物标志物已被报道，但没有一个得到充分验证 （例如，FDA 批准）。这主要是因为缺乏提供准确、准确的分析方法。 对生物样本中极低水平的 microRNA 进行可重复且经济高效的定量。 当前 microRNA 检测的黄金标准（即定量逆转录聚合酶链反应， RT-qPCR）提供“相对定量”，并且检测成本非常高（每次检测消耗品 > 15 美元）。 毕竟，所有基于 PCR 的定量分析都部署了在荧光信号之间建立的校准曲线 以及 microRNA 浓度的对数（而不是 microRNA 浓度），它本质上产生 定量结果不太准确，并以指数方式放大荧光信号中包含的不确定性 该研究的目标是消除目前 microRNA 定量分析的局限性， 从而促进这些新兴疾病生物标志物的生物医学研究和全面验证。 本文提出了一种基于稳健且流行的方法对目标 microRNA 进行“绝对定量”的新颖分析策略 超高效液相色谱-串联质谱 (UPLC-MS/MS) 与亲和力相结合 磁性固相萃取和等温信号放大该策略涉及以下 PCR-。 免费工作流程：1) 使用 ssDNA 探针从生物样品中提取/富集目标 microRNA - 磁珠缀合物；2) 聚腺苷酸聚合酶捕获的目标 microRNA 的固体表面聚腺苷酸化 13C-标记的腺嘌呤；和3)酸水解后13C-标记的腺嘌呤的UPLC-MS/MS测定 扩展靶点 microRNA 预计具有以下检测特征：高 灵敏度（LOQ < 1pM，生理相关水平）、高精度（回收率 ≥ 95%）、良好的重复性 (RSD ≤ 5%)，单碱基错配辨别能力，检测中无需分离总RNA， 和成本效益（每次检测的耗材总成本 < 1.5 美元。实施该分析方法 将对microRNA生物医学研究产生深远影响。 拟议的项目适合杰克逊州立大学的研究重点，并将能够吸引 化学和生物课程的学生将获得实验室技能，包括 细胞培养、生物样品制备、仪器分析和科学数据处理/演示 总的来说，该项目将有助于发展和维持 JSU 的卓越研究。 （HBCU），从而为我们未来研究队伍的多样性做出贡献。