Abstract 4205: Simultaneous high resolution fluorescence imaging of cellular DNA and RNA enabled by complex oligonucleotide libraries in various sample types.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Fluorescence in situ Hybridization (FISH) is a powerful technique for determining the structure, organization, and localization of specific nucleic acid sequences within individual cells. However, the use of FISH has often been dependent upon access to cloned template DNA for the generation of probes, which can be difficult if clones for specific regions are unavailable, or if the genomic region of interest contains repetitive and/or other problematic sequences. We have leveraged our ability to chemically synthesize DNA in massively parallel reactions to produce libraries of oligonucleotides up to 200 bases in length that can be used for the generation of FISH probes. The sequences of the oligonucleotides in these libraries are selected in silico using empirically determined criteria so as to avoid repetitive elements or regions homologous to other non-targeted loci. Using oligonucleotide library-derived FISH probes on DNA, human genomic regions as small as 1.8 kb and as large as whole chromosomes can be visualized in both metaphase and interphase cells using the same simple assay protocol. Because of the inherent flexibility in our probe design methods, we readily visualized regions rich in repeats and/or GC content. We have also used these oligonucleotide library-derived FISH probes to detect the localization of a variety of both coding and non-coding RNAs in fixed cells, using both conventional fluorescence and structured illumination microscopy. Simultaneous hybridization of FISH probes labeled with different fluorophores enables visualization of multiple sequences at once. Using probes designed specifically to transcribed vs. non-transcribed regions, we have been able to simultaneously detect DNA and RNA from the same locus in the same FISH assay. We have successfully used this technique in several types of fixed tissue culture cells as well as in formalin-fixed paraffin-embedded tissue sections. Our oligo FISH methods are readily compatible with the co-detection of cellular proteins by immunocytochemistry. The ability to generate high performance FISH probes using chemically synthesized oligo libraries that can work flexibly with co-detection of other molecules yields a valuable tool for studies of how localization of specific nucleic acids impacts biological function. Citation Format: Robert A. Ach, Peter Tsang, Alicia Scheffer-Wong, Laurakay Bruhn, Weston Powell, Janine LaSalle, Alice Yamada. Simultaneous high resolution fluorescence imaging of cellular DNA and RNA enabled by complex oligonucleotide libraries in various sample types. [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 4205. doi:10.1158/1538-7445.AM2013-4205