© Copyright 2017 Alison Marie Thompson Single-Cell Molecular Profiling of Nucleic Acids in the Microfluidic Self- Digitization Chip Alison Marie Thompson A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2017 Reading Committee: Daniel T. Chiu, Chair Tomikazu Sasaki Frantisek Tureček Program Authorized to Offer Degree: Chemistry University of Washington Abstract Single-Cell Molecular Profiling of Nucleic Acids in the Microfluidic Self- Digitization Chip Alison M. Thompson Chair of the Supervisory Committee: Professor Daniel T. Chiu Chemistry 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. i 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. ii TABLE OF CONTENTS List of Tables ............................................................................................................................... viii Chapter 1. Introduction ................................................................................................................... 1 1.1 Overview ......................................................................................................................... 2 1.2 Introduction to PCR Techniques ..................................................................................... 4 1.2.1 PCR Amplification...................................................................................................... 5 1.2.2 PCR Detection Chemistries ........................................................................................ 7 1.2.3 Real-Time PCR and Digital PCR ............................................................................... 8 Chapter 2. Review of Microfluidics for Single-Cell Genetic Analysis ........................................ 12 2.1 Abstract ......................................................................................................................... 13 2.2 Introduction ................................................................................................................... 13 2.3 Microfluidics as a solution ............................................................................................ 18 2.4 Capture and enrichment of single cells ......................................................................... 19 2.5 Compartmentalization ................................................................................................... 22 2.6 Analysis of single-cell genetic material ........................................................................ 25 2.7 Future outlook ............................................................................................................... 31 Chapter 3. The Self-Digitization Microfluidic Chip for the Absolute Quantification of mRNA in Single Cells ................................................................................................................................... 33 3.1 Abstract ......................................................................................................................... 34 3.2 Introduction ................................................................................................................... 35 3.3 Methods......................................................................................................................... 37 3.3.1 Single-cell qPCR ....................................................................................................... 37 3.3.2 Microfluidic Device Fabrication. .............................................................................. 37 3.3.3 Device Loading ......................................................................................................... 37 3.3.4 Digital RT-PCR. ....................................................................................................... 39 3.3.5 Data processing. ........................................................................................................ 40 3.3.6 RNA standard curve. ................................................................................................. 41 iii 3.3.7 Single Cell mRNA FISH. ......................................................................................... 41 3.3.8 Cell Culture. .............................................................................................................. 42 3.4 Results and Discussion ................................................................................................. 42 3.5 Conclusions ................................................................................................................... 52 3.6 Supplemental Information ............................................................................................ 53 Chapter 4. Single-Cell Isolation and One-step genotyping in the microfluidic self-digitization chip ................................................................................................................................................ 60 4.1 Abstract ......................................................................................................................... 61 4.2 Introduction ................................................................................................................... 62 4.2.1 Genetic Heterogeniety (Clonality) in Cancer ........................................................... 62 4.2.2 Clonality in the Bulk ................................................................................................. 63 4.2.3 Single-Cell Genetic Approaches ............................................................................... 64 4.3 Materials and Methods .................................................................................................. 65 4.3.1 Cell Lines and Template DNA ................................................................................. 65 4.3.2 Allele Discrimination Primers and Probes ................................................................ 66 4.3.3 PCR Conditions ........................................................................................................ 67 4.3.4 Cell lysis experiments ............................................................................................... 67 4.3.5 SD Chip Fabrication ................................................................................................. 68 4.3.6 SD Chip Priming and Loading .................................................................................. 68 4.3.7 Imaging of Captured Cells ........................................................................................ 69 4.3.8 SD Chip thermalcycling ............................................................................................ 70 4.3.9 Post-PCR Imaging .................................................................................................... 70 4.3.10 SD Chip Image analysis ........................................................................................ 70 4.4 Results and Discussion ................................................................................................. 71 4.4.1 Cell lysis.................................................................................................................... 71 4.4.2 Characterization of Pre-Made PCR Master Mixes ................................................... 74 4.4.3 Detection Chemistries for