Microfluidic chromatin immunoprecipitation
Abstract/Contents
- Abstract
- Protein--DNA interactions are responsible for numerous critical cellular events: for example, gene expression and silencing are mediated by transcription factor protein binding and histone protein modifications, and DNA replication and repair rely on site specific protein binding. Chromatin immunoprecipitation (ChIP) is the only molecular assay that directly determines, in a living cell, the binding association between a protein of interest and specific genomic loci. It is an indispensible tool in the biologists' toolbox, but the many limitations of this technique prevent broad adoption of ChIP in biological studies. The typical ChIP assay can take up to one week to complete, and the process is technically tricky, yet tedious. ChIP assay yields are also low, thus requiring on the order of millions to billions of cells as starting material, which makes the assay unfeasible for studies on rare or precious samples. Cancer stem cells (CSCs), for instance, are obtained from primary tumors, and FACS sorting of the dissociated tumor cells rarely yields more than ~100,000 CSCs per tumor. This thesis describes the microfluidics-based strategies for performing ChIP. The first design for a microfluidic ChIP design utilizes the automation and scalability aspects of microfluidics to reduce both total and hands-on assay time, and improve throughput. It can take chromatin prepared using any existing ChIP protocol as input, and generate ChIP-qPCR results in just 1 day. The device is shown to be comparable to existing ChIP protocols, and can detect cellular epigenetic changes induced by external cytokine stimulants. It was used to investigate transcription factor binding dynamics in an aging-related pathway, and was able to link transcription factor binding to local changes in histone modifications. The second design of the microfluidic ChIP platform focuses on addressing the cell number requirements of ChIP. In addition to reducing assay time through automation, this design incorporates microfluidic designs that allow whole fixed cells as input, and enables automated ChIP from as few as 2,000 cells. Finally, using this high-sensitivity ChIP device in conjunction with next-generation sequencing technology, a protocol for determining genome-wide epigenetic landscapes from as few as 2,000 cells is developed, with the ultimate goal of performing protein-DNA association studies on CSCs.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2012 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Wu, Angela Ruohao |
|---|---|
| Associated with | Stanford University, Department of Bioengineering. |
| Primary advisor | Quake, Stephen Ronald |
| Thesis advisor | Quake, Stephen Ronald |
| Thesis advisor | Chang, Howard |
| Thesis advisor | Felsher, Dean (Dean Walton) |
| Advisor | Chang, Howard |
| Advisor | Felsher, Dean (Dean Walton) |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Angela Ruohao Wu. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2012. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Angela Ruohao Wu
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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