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Microfluidic technology for high-throughput single cell gene expression analysis

University of British Columbia

Transcription measurements with single cell resolution are critical to understanding variable responses in immunity, measuring stochastic noise in gene expression, and assessing the disease and developmental state of heterogeneous populations. The latter is particularly important in stem cell science, developmental biology, and cancer, where minority cells may be most significant. To see these populations requires the quick and cost-effective measurement of hundreds to thousands of individual cells. Quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for quantitative analysis of transcript levels that provides excellent sensitivity and dynamic range in the detection of transcripts. However, the use of RT-qPCR is generally limited to ensemble measurements of bulk cells or plasma, and is blind to minority cell populations. This aggregation obscures the underlying biological response and variability. To address this limitation, we exploit recent advances in scalable microfluidics to develop robust lab-on-chip technology capable of highly parallel and cost-effective measurements of transcript levels from single cells. The microfluidic device integrates single-cell capture, lysis, reverse transcription of contained RNA, and precise measurement of cDNA using RT-qPCR. We demonstrate this system in the study of microRNA expression in a cell line representing chronic myelogenous leaukemia, pluripotency markers in differentiating human embryonic stem cells, and the detection of somatic mutations in a primary breast cancer sample. The ability to screen isolated cells by simultaneously measuring the fraction of cells expressing a specific gene and quantifying the abundance of expression, may provide a new modality for the early detection of disease such as cancer.

Thesis/Dissertation

eng

Vancouver : University of British Columbia Library

10.14288/1.0071211

Master of Applied Science - MASc

Applied Science, Faculty of

2010-11

Graduate

DSpace