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

基于无 PCR UPLC-MS/MS 的 microRNA 定量分析

基本信息

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
项目类别：
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

项目摘要

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.

项目总结