Rapid and high-specificity assay for micro-RNA detection using combined on-chip isotachophoresis and affinity hydrogel purification
Abstract/Contents
- Abstract
- microRNAs are short, non-coding RNA molecules that regulate gene expression in animal and plant cells and play important roles in diverse cellular functions. Dysregulation of microRNAs has been linked to diseases such as diabetes, alzheimer's disease, and many forms of cancer. microRNA expression signatures have therefore emerged as important new clinical biomarkers with both diagnostic and prognostic value. Despite growing interest, quantitative microRNAs continues to pose challenges for traditional RNA profiling methods due to their low abundance, high degree of sequence similarity, and complex biogenesis. In this work, we demonstrate the development of a microfluidic microRNA detection assay which addresses these challenges by leveraging two key technologies: on-chip isotachophoresis (ITP) and photopatterned functionalized hydrogels. ITP is a robust electrokinetic technique which uses a heterogeneous buffer system to perform greater than 10,000-fold focusing. In addition to providing signal enhancement, ITP focusing also accelerates reaction kinetics of nucleic acid hybridization. Our microRNA detection assay first uses ITP to enhance hybridization between microRNAs and complementary DNA reporters. Following hybridization, the ITP zone migrates into a purification region which a hydrogel decorated with DNA capture probes. Excess (unhybridized) reporters bind to the capture probes and become immobilized, while reporters hybridized to microRNAs remain focused in ITP and can be detected downstream. In the first part of the dissertation, we present a proof of concept study to assess the feasibility of a microRNA assay and characterize assay sensitivity and dynamic range under low stringency conditions. We demonstrate our technique performs quantitative analysis of synthetic microRNAs with 4 orders of magnitude dynamic range, near 1 pM (30,000 molecules) sensitivity, and assay run time of only 10 min. This constitutes a 100- fold improvement in dynamic range and limit of detection over previous ITP-based approaches. In the second part of the dissertation, we present modifications to our basic assay which allow us to detect microRNAs with single-nucleotide specificity (high stringency) and using total RNA extracted from real tissues. Using this modified assay, we quantify concentration of let-7a microRNA in mouse and human tissues, and validate these measurements, and our method more generally, by comparison with qPCR. We further develop a numerical model which demonstrates that our modified assay leverages both thermodynamics (in the first stage) and off-rate kinetics (in the second stage) to enhance hybridization specificity.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2013 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Garcia, Giancarlo | |
|---|---|---|
| Associated with | Stanford University, Department of Mechanical Engineering. | |
| Primary advisor | Santiago, Juan G | |
| Thesis advisor | Santiago, Juan G | |
| Thesis advisor | Mani, Ali, (Professor of mechanical engineering) | |
| Thesis advisor | Pruitt, Beth | |
| Advisor | Mani, Ali, (Professor of mechanical engineering) | |
| Advisor | Pruitt, Beth |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Giancarlo Garcia. |
|---|---|
| Note | Submitted to the Department of Mechanical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2013. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2013 by Giancarlo Garcia
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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