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

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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.