Single-Cell Molecular Profiling of Nucleic Acids in the Microfluidic Self-Digitization Chip

Abstract

In the last four decades, advancements in technologies to copy, measure, and manipulate DNA and RNA in situ have enabled new methods for research and diagnosis of disease. As a subset of these methods, novel microfluidic platforms have emerged that have demonstrated assay that can run at lower-cost, have lower-sample consumption, are more robust, are easier-to-perform, and/or are more accurate. For genetic analyses, microfluidic technologies have been used to integrate DNA and RNA measurement with upstream sample handling, such as single cell manipulation. Performing genetic analysis on single cells can uncover cell-to-cell variation, or heterogeneity, masked by measurements on homogenized tissues. This heterogeneity has implications for organism development and disease. Intercellular heterogeneity is thought to play a critical role in cancer, where subsets of a population of cancerous cells can seed metastasis, evade the immune system, evade therapies, or cause relapse post-treatment. In this thesis, methods to perform single-cell genetic measurements are described using a simple microfluidic device, the Self-Digitization Chip (SD Chip). An overview of microfluidics for single-cell genetic analysis is provided, highlighting the advantages of performing analyses at nL scale platforms over µL- or mL-scale platforms. A method is described to quantify absolutely the amount of a specific mRNA species in a single cell by digital RT-PCR in a one-step reaction. The quantities of mRNA measured in a single cell are found to compare well to values obtained by mRNA fluorescence in situ hybridization (FISH). This is the first study to show absolute quantification of mRNA by digital PCR. A single-cell genotyping method is also described. This method is used for genotyping of single cells to determine zygosity of a gene of interest for acute myeloid leukemia. The device allows for genotyping of hundreds of single cells individually in a single PCR run. The reaction chambers are stationary throughout imaging and PCR, allowing us the ability to quantify and eliminate from our zygosity calls measurement errors such as false negatives and false positives. Possible future directions for the SD Chip are also discussed, including work on unprocessed, whole-blood samples.