Abstract 361: Single cell detection of protein and RNA: Development of a novel method combining immunocytochemistry and in situ hybridization in breast cancer cells

Immunocytochemistry (ICC) utilizes antibodies to visualize the localization of specific proteins in cells while in situ hybridization (ISH) is a useful research tool for examining the presence and changes in levels of gene expression in cells. The ability to simultaneously visualize the co-localization of protein and RNA species in individual cells is necessary to address important biological questions. Additionally, studying both protein and RNA can be useful in cases where expression levels vary between mRNA and protein and to detect post-translational modifications. However, a combined technique has proved challenging due to the incompatibility of ICC and ISH protocols. Furthermore, traditional ISH methods have lacked sensitivity to detect low abundance RNA species and have limited multiplexing capability. Here, we describe a novel technique combining ICC staining with RNA ISH (based on the ViewRNA Assay), which utilizes branched DNA signal amplification technology allowing for single copy mRNA sensitivity. We demonstrate the utility and versatility of this technique using a variety of antibody and probe combinations relevant for cancer research. We compared expression of protein and mRNA of HER2, estrogen receptor alpha, and progesterone receptor in several breast cancer cell lines (MCF7, SKBR3, MDA-MB-175, MDA-MB-231, and BT474) known to express differential levels of these targets. We also compared expression of several microRNAs (let-7a, miR-17, miR-30d, miR-107, and miR-93) known to play a role in cancer development in combination with staining for proteins involved in proliferation (Ki-67), anti-apoptosis (survivin), and cell structure (tubulin). We show successful detection of low abundance RNA species together with various protein markers. This novel technique is a valuable tool not only for cancer researchers, but also for investigators across numerous fields in biological research. Citation Format: Lisa J. Ma, Richard T. Frank, Xiaoxiao Li, Steve Lai, Jeanette Schmidt, Peggy Just, Sara Becker-Catania. Single cell detection of protein and RNA: Development of a novel method combining immunocytochemistry and in situ hybridization in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 361.