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

项目摘要 已发现microRNAs的异常表达与病理条件有关。过去的几年里 已经报道了许多基于miRNA的疾病生物标志物，但没有一个得到完全验证。 (例如，用于FDA批准)。这在很大程度上是因为缺乏一种分析方法来提供准确、 对生物样本中存在的非常低水平的microRNA进行可重复的、成本效益高的定量。这个 目前用于microRNA检测的金标准(即定量逆转录聚合酶链式反应， RT-qPCR)提供了“相对定量”，而且检测成本非常高(&gt；每份检测耗材15美元)。 毕竟，所有基于PCR的定量分析都采用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)，它在自然界中产生 较不准确的定量结果，并以指数方式放大荧光信号中包含的不确定性 测量。这项研究的目标是消除目前MicroRNAs定量分析的局限性， 从而促进了对这些新出现的疾病生物标志物的生物医学研究和充分验证。我们建议 本文提出了一种新的基于稳健性和普及性的靶向microRNAs绝对定量分析策略 超高效液相色谱-串联质谱法与亲和力联用 磁性固相萃取和等温信号放大。该策略涉及以下聚合酶链式反应- 免费工作流程：1)通过使用单链DNA探针从生物样本中提取/富集靶向microRNA- 磁珠偶联物；2)Poly(A)聚合酶捕获的靶microRNA的固体表面多聚腺苷 用~(13)C标记的腺嘌呤；3)酸水解后的UPLC-MS/MS测定~(13)C标记的腺嘌呤 延伸的靶标microRNA。预计定量检测将具有以下检测特征：高 灵敏度(LOQ&lt；1 PM，生理相关水平)、高精度(回收率≥为95%)、良好的重复性 (RSDRNA5%)，单碱基错配识别能力，不需要总≤提取， 和成本效益(耗材的总成本&lt；每份化验1.5美元。这一分析方法的实施 将对microRNA生物医学研究产生深远影响。 提出的项目符合杰克逊州立大学的研究重点，并将能够从 化学和生物专业的学生。参与的学生将获得实验技能，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 通过研究活动。总的来说，该项目将有助于发展和保持联合苏黎世大学的卓越研究成果。 (HBCU)，从而有助于我们未来研究人员的多样性。

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.