The ability to visualize both RNA and protein expression in a single formalin-fixed paraffin-embedded (FFPE) tissue section is a powerful technique for studying biological problems such as the regulation of protein expression and RNA/protein interactions, as well as for antibody characterization and validation. We have leveraged our ability to chemically synthesize oligonucleotides in massively parallel reactions to produce DNA libraries that can be used to generate probes for the detection of DNA or RNA by Fluorescence in situ Hybridization (FISH). 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 nucleic acids. We have used these oligonucleotide library-derived FISH probes in combination with a novel signal amplification technique to detect the localization of a variety of both coding and non-coding RNAs in fixed tissue culture cells and FFPE tissue sections, using both conventional fluorescence and structured illumination microscopy. We report here combining our RNA FISH methodology with antibody-based fluorescent immunohistochemistry (IHC) detection of proteins on the same FFPE slide. We show that our combined RNA FISH/IHC protocol works on a variety of RNA probe/antibody combinations for both nuclear and cytoplasmic proteins. We also show that our fluorescent assay can be converted to a colorimetric one for both the RNA and protein detection.

Citation Format: Robert A. Ach, May Tom-Moy, Peter Tsang, Kelly Kroeger. Combining oligonucleotide library-based RNA fluorescence in situ hybridization and fluorescent immunohistochemistry on the same tissue section [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1403.