abstract

November 21, 2003

 

Simultaneous Analysis of Gene Expression in Single Cells
Sheri J. Lillard, UC Riverside

The goal of this research is to understand fundamental cell biology involved with transcription and translation in single cells. Most gene expression studies in mass-limited samples involve the analysis of either the mRNA or the protein. However, there is often a disparity between the levels of mRNA and protein from the same sample. In other words, simply calculating protein expression, based on mRNA analysis, is insufficient since protein levels can vary substantially for a given mRNA copy number. Similarly, only measuring protein abundance might not reveal important transcriptional information as related to expression. Thus, an accurate understanding of expression at the molecular level requires knowing the levels of both the transcript and the protein. Furthermore, the expression levels of mRNA and protein represented by a population average do not accurately reflect intercellular variation that is known to be present. Monitoring intercellular variation is only possible with a method capable of single-cell distinction. Our electrophoretic methods are well suited for the analysis of individual cells, primarily owing to ultrasensitive detection schemes and single-cell sample-handling capabilities. We have demonstrated the ability to detect b-actin, as well as estrogen receptor-in individual mammalian cells using a combination of reverse transcriptase-polymerase chain reaction (RT-PCR) and capillary electrophoresis with laser induced fluorescence (CE-LIF). Furthermore, we have achieved preliminary results in monitoring b-actin protein using a fluorescent complexation reaction. Finally, we have established a protocol by which nucleic acid can be selectively isolated from nL-sized samples-a critical part of the interface to achieve simultaneous analysis.