Single cell mRNA analysis is becoming increasingly important as a tool to understand gene regulation, cell differentiation and the development of disease states. For example, in stem-cell research, important questions are: How does cell differentiation alter the gene expression profiles? What is the sequence of gene activations that ultimately leads to a change in cell lineage? Such questions can only be addressed by observing gene expression at the single cell level because of the heterogeneous nature of cell populations. This proposal will address the fundamental problem of efficiently capturing mRNA from individual single eukaryotic cells and processing them at high throughput using a droplet microfluidics device. Droplet microfluidics in which samples and reagents are encapsulated in aqueous microdroplets (1 pL- 10 nL) dispersed in a fluorocarbon oil carrier is particularly suited for single-cell manipulation. Among many advantages of the droplet format: 1- contamination is prevented by the physical and chemical isolation of the droplets from each other and the walls of the devices;2- droplets can be fully manipulated and easily retrieved without any moving part or elaborate automation at speeds up to 3000 droplets/sec;3- this technique has been already shown to be compatible with molecular biology techniques such as nucleic acid amplification by polymerase chain reaction. Despite these advantages, it has so far been difficult to perform multi-step reactions involving buffer exchanges using this technique because of the lack of methods to continuously extract and concentrate molecules of interest within individual droplets. The main innovation of this proposal is the development and characterization of a novel method to extract and concentrate mRNA molecules that are bound to magnetic microparticles inside aqueous droplets. Extraction and concentration is achieved by splitting droplets along the droplet flow axis under the influence of a magnetic field to separate the part of the droplet that contains microparticles from the part that is devoid of particles. In particular, we propose to develop a workflow performing the subsequent steps: (a) Encapsulation of single cells into droplets;(b) Cell lysis inside droplets;(c) Extraction of mRNA using Oligo (dT) functionalized magnetic microbeads with subsequent concentration and buffer changing steps;(d) First strand cDNA synthesis on microparticle bound mRNA. This method yields emulsion libraries with each droplet containing the whole cDNA collection of a single-cell bound to magnetic particles.
The results of this proposal will enable the development of high throughput methods to measure single cell gene expression. Such technology is urgently needed for monitoring and evaluating cancer treatment and as a basic science tool to study cell differentiation of stem cells and cancer progression.
|Brouzes, Eric; Kruse, Travis; Kimmerling, Robert et al. (2015) Rapid and continuous magnetic separation in droplet microfluidic devices. Lab Chip 15:908-19|
|Brouzes, Eric (2012) Droplet microfluidics for single-cell analysis. Methods Mol Biol 853:105-39|