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

项目摘要 已经发现microRNA的异常表达与病理状况相关。在过去数年里 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 (e.g., FDA批准）。这主要是因为缺乏一种分析方法， 可重复的，并且具有成本效益的定量在生物样本中以非常低的水平存在的microRNA。的 目前用于微小RNA测定的金标准（即，定量逆转录聚合酶链反应， RT-qPCR）提供"相对定量"，并且测定成本非常高（消耗品/每次测定>$15）。 毕竟，所有基于PCR的定量测定都部署了在荧光信号之间建立的校准曲线， 和microRNA浓度的对数（而不是microRNA浓度），其自然产生 不太准确的定量结果，并指数放大了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对microRNA定量分析的限制， 从而促进生物医学研究和这些新兴疾病生物标志物的充分验证。我们提出 在此，基于稳健和流行的用于靶微小RNA的"绝对定量"的新的分析策略 超高效液相色谱-串联质谱法（UPLC-MS/MS）结合亲和层析 磁性固相萃取和等温信号放大。该战略涉及以下PCR- 自由的工作流程：1）通过使用ssDNA探针从生物样品中提取/富集靶microRNA- 磁珠缀合物; 2）由poly（A）聚合酶捕获的靶microRNA的固体表面聚腺苷酸化 用13C-标记的腺嘌呤;和3）UPLC-MS/MS测定13C-标记的腺嘌呤的酸水解后的13C-标记的腺嘌呤 延伸的靶向微小RNA。预期定量测定具有以下测定特征： 灵敏度（LOQ <1pM，生理相关水平），准确度高（回收率≥ 95%），重复性好 (RSD≤ 5%），单碱基错配辨别能力，在测定中不需要总RNA分离， 和成本效益（每次化验消耗品的总成本<$1.5）。本分析方法的实施 将对microRNA生物医学研究产生深远的影响。 该项目符合杰克逊州立大学的研究重点，将能够绘制 化学和生物学专业的学生。参与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 通过研究活动。总的来说，该项目将有助于发展和维持JSU的卓越研究 (an HBCU），从而有助于我们未来的研究队伍的多样性。

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.