New technologies for single-cell RNA expression profiling have transformed our understanding of cell biology. Despite several variations, the vast majority of available methods are applicable only to messenger RNA expression measurements, leaving microRNAs and other classes of small RNAs largely unexplored. In this dissertation I describe the creation and application of technology that allows for the efficient and precise analysis of microRNA expression in large numbers of single cells.
First, the foundational components necessary for microfluidic integration of single-cell RT-qPCR analysis were developed. The resulting device executes and parallelizes all steps of cell capture, cell lysis, reverse transcription, and quantitative PCR on up to 300 cells per run. In comparison to standard benchtop assays, nanolitre-volume processing was found to increase measurement performance on samples with limited template. The core functionality established in this part of the work provides a foundation for further microfluidic single-cell assays.
The capabilities of this platform were next expanded to enable highly multiplexed RT-qPCR analysis. By incorporating sophisticated microfluidic fabrication techniques with an extended workflow that included a pre-amplification step, the number of assays per cell was increased from one or two to up to forty, while maintaining the same benefits to measurement performance that were previously observed.
Finally, small-volume analysis and carefully optimized molecular biology were combined to develop a method to generate high-quality single-cell microRNA sequencing libraries. This method was then applied to provide a comprehensive look at microRNA expression dynamics across the human hematopoietic cell hierarchy. The results indicated that the population structure derived from miRNA expression profiles supported a model of continuous, linear hematopoietic stem cell differentiation, in contrast to the prevailing model of stepwise, branched lineage commitment. An expanded set of miRNA markers that are highly expressed in HSCs, decrease gradually during differentiation, and are absent in mature cells were also identified. Finally, an analysis of the relative expression of microRNA isoforms was performed, showing that they are a dynamic feature that varies between different microRNAs and cell types.
The capabilities conferred by this suite of microfluidic devices will enable the continued, routine analysis of microRNA expression in single cells.Science, Faculty ofGraduat
