Abstract LB-240: A novel approach to detecting RNA expression in living breast cancer cells.

Abstract

Abstract When studying the complexities of cancer biomarkers, the ability to monitor gene expression within live cells that have become cancerous provides better insight than examining fixed or lysed tissues. However, monitoring RNA within intact cells can prove to be challenging with current techniques, which involve complex RNA isolation procedures that compromise cell viability. Further, amplification methods can create false positives or erroneously inflate differences. Here we describe a probe-based detection method that allows for the detection of ErbB-2 and EGFR mRNA levels across live intact SK-BR-3 breast cancer cells without the need for transfection reagents. The detection methodology is based upon an oligonucleotide gold nanoparticle conjugate capable of detecting intracellular levels of mRNA and miRNA in living cells. The probes enter the cell by means of the cell's native endocytosis machinery. Upon binding to the complementary target RNA sequence, a fluorescent signal (flare) is released, which allows the cells to be detected on any fluorescence analysis platform. In contrast to traditional RNA detection methods, since our technique allows for detection within live cells without altering their gene expression, the same cells that were profiled for EGFR and ErbB-2 expression could be sorted based on their specific mRNA content and subsequently further studied. Stimulation or treatments could then be performed on the previously profiled cells and assessed using probes for additional RNA markers or more traditional techniques such as antibody staining, qRT-PCR, or Array profiling. For this purpose, we combined immunostaining for cell surface EGFR with fluorogenic nanoparticle-based detection of EGFR mRNA in 4 breast cancer cell lines known to range in EGFR expression levels, and observed significant correlation for the signals in two-color flow cytometry. Thus, detecting RNA expression levels in live cancer cells with the ability to utilize the same cells in downstream testing gives researchers the ability to perform experiments which were previously thought to be impossible. Citation Format: Don Weldon, Kevin Su, Alex Ko, Grace Johnston, Matthew Hsu, Lucas Armstrong. A novel approach to detecting RNA expression in living breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-240. doi:10.1158/1538-7445.AM2013-LB-240