School of Electrical, Computer and Energy Engineering
PhD Final Oral Defense Single Cell RT-qPCR on 3D Spheroid Cells
by Kuo-chen Wang 11/03/2016 09:00AM BDA250
Committee: Dr. Deirdre R. Meldrum (chair) Dr. Shih-hui Chao Dr. Hong Wang Dr. Michael Goryll
Abstract
A single cell is the very fundamental element in an organism; however, it contains the most complicated and stochastic information, such as DNA, RNA, and protein expression. It is a necessity to study the stochastic gene expression in order to discover the biosignature at the single-cell level. The heterogeneity gene expression of single cells from an isogenic cell population has already been studied for years. Yet to date, single- cell studies have been confined in a fashion of analyzing isolated single cells or dilution cells from the bulk-cell populations. These techniques or devices are limited by either the mechanism of cell lysis or the difficulties to target specific cells without harming neighboring cells.
This dissertation presents a development of a laser lysis chip combined with a two- photon laser system to perform single-cell lysis and two-step reverse transcription- quantitative polymerase chain reaction (RT-qPCR) analysis in situ three-dimensional
(3D) cell clusters. The well-designed laser lysis chip can be used to trap 3D spheroid cells
or tissue like samples. By utilizing a two-photon laser, each single cell can be identified and lysed from one to another accordingly in 3D spheroid cells. During the lysing process, the contents inside the target cell will be released into surrounding media and carried out to the lysate collectors. By conducting the two-step RT-qPCR, the gene expression of each individual cell can be analyzed and traced back to its original location on spheroid cells. Using this system, single-cell studies can be approached to not only single separated cells, but intact 3D spheroid cells or even tissue samples, which can be helpful to study the prognosis of premalignant conditions and identify early tumor development.