Development of methodology for the study of nucleic acids from single cells using capillary electrophoresis

The work presented herein describes the development of capillary electrophoresis (CE)-based methodology for the analysis of nucleic acids originating from individual cells. Three distinct approaches were used to accomplish this goal: (1) evaluation of nucleic acid dyes for DNA detection; (2) the study of DNA and RNA migration in the presence of electroosmotic flow; and (3) the development of a method to measure single-cell messenger RNA (mRNA) using the reverse transcriptase-polymerase chain reaction (RT-PCR).; Initial studies involved the examination of nucleic acid dyes, in conjunction with laser-induced fluorescence (LIF) detection, for the visualization of DNA restriction fragments and single-cell RNA. Specifically, SBYR Gold and SYBR Green I were used to detect RNA from single cells or a double-stranded (ds)DNA ladder following CE. Both 275- and 488-nm excitation sources were assessed. Spectral characterization of each dye was performed, followed by a detailed comparison of the detection sensitivity for dsDNA and RNA samples.; In the next phase, a method to monitor DNA and RNA migration in the presence of electroosmotic flow (EOF) was developed. As a consequence of their high negative charge density, the free-solution electrophoretic mobility of nucleic acids opposes EOF. Thus under conditions of moderate EOF, DNA may be selectively ejected from a capillary into an aliquot of water. This phenomenon was initially applied to the co-detection of protein and RNA from cell lysate samples, as EOF retains positively-charged species within the capillary. The ejected RNA was analyzed on a separate CE-LIF system. In later studies, a detailed characterization of this process was performed using DNA restriction fragments and the dsDNA product of the single-cell RT-PCR.; Finally, an approach was established to evaluate single-locus and multiplex gene expression of individual cells using RT-PCR and CE-LIF. Preliminary studies involved the development of the RT-PCR protocol and examination of cell lysis procedures for the measurement of single-cell beta-actin mRNA. Products were visualized using a hydroxypropylmethylcellulose sieving matrix containing ethidium bromide. This method was subsequently optimized and applied to the simultaneous amplification of beta-actin and estrogen receptor alpha genes, representing the first demonstration of the detection of single-cell multiplex products by CE.