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.

项目摘要 已经发现microRNA的异常表达与病理条件相关。在过去的几年中 已经报道了许多基于MiRNA的假定基于miRNA的疾病生物标志物，但均未得到充分验证 （例如，用于FDA批准）。这主要是因为缺乏提供准确的分析方法 在生物样品中，可重复的microRNA可重复和具有成本效益的量化。 当前的MicroRNA测定的黄金标准（即定量逆转录聚合酶链反应， RT-QPCR）提供“相对数量”，并且测定成本非常高（每种测定量> $ 15）。 毕竟，所有基于PCR的定量测定都在荧光信号之间部署校准曲线 和microRNA浓度的对数（而不是microRNA浓度），本质上会产生 精确的定量结果和指数放大器荧光信号中包含的不确定性 测量。该研究的目的是消除当前对microRNA的定量评估的局限性， 这是对这些新兴疾病生物标志物的生物医学研究和全面验证。我们建议 以下是基于强大而流行的目标microRNA的“绝对量化”的新分析策略 超出性液相色谱串联质谱法（UPLC-MS/MS）与亲和力结合 磁性固相提取和等温信号扩增。该策略涉及以下PCR- 自由工作流程：1）通过使用ssDNA探针从生物样品中提取 /富集目标microRNA 磁珠结合； 2）聚（A）聚合酶捕获的靶microRNA的固体表面聚腺苷酸化 带有13c标记的腺嘌呤； 3）在酸水解后，UPLC-MS/MS测定13C标记的腺嘌呤 扩展目标microRNA。定量测定有望具有以下测定特征：高 敏感性（LOQ <1pm，具有物理相关水平），高精度（恢复≥95％），良好的可重复性 （RSD≤5％），单基碱基不匹配歧视的能力，无需在测定中进行总RNA隔离， 和成本效益（每个测定的消耗品总成本<$ 1.5。实施此分析方法 将对microRNA生物医学研究产生深远的影响。 该项目提出的适合杰克逊州立大学的研究集中 化学和生物学计划的学生。参加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过程 /呈现 通过研究活动。总的来说，该项目将有助于开发和维持JSU的卓越研究 （HBCU），因此有助于我们未来的研究劳动力的多样性。