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79 Multiplexed detection of RNA and protein to interrogate the tumor-immune landscape with a novel automated RNAscope™ assay
  1. Sonali Deshpande1,
  2. Anushka Dikshit2,
  3. Ge-Ah Kim1,
  4. Julia Yu1,
  5. Laetitia Chatelain1,
  6. Nancy George1,
  7. Li-Chong Wang2 and
  8. Maithreyan Srinivasan1
  1. 1Advanced Cell Diagnostics, a Bio-Techne brand, Newark, CA, USA
  2. 2Advanced Cell Diagnostics, Minneapolis, MN, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Studying cell-cell interactions can have important implications in immuno-oncology, inflammation, and neuroscience. Tissue heterogeneity poses immense challenges to understanding underlying molecular mechanisms using techniques such as qRT-PCR or bulk sequencing. While single-cell RNA sequencing can provide information about precise cellular composition of tissues, spatial context is lost. With platforms such as RNAscope, target gene and protein expression can be visualized to characterize cell types and tissue neighborhoods. Here, we demonstrate a novel method for the simultaneous detection of RNA and protein using a modified co-detection assay.

Methods This novel co-detection assay enables visualization of a combination of up to 12 RNA and/or protein targets on the same sample. We used a set of antibodies targeting key immune and tumor cell markers- PD1, CD3, CD4, CD8, CD68, FOXP3 and KRT17, along with RNA biomarkers to interrogate the tumor microenvironment (TME) in human FFPE tumor samples.

Results Using a combination of RNA and protein targets, we characterized different cell types such as T cells, macrophages, and tumor cells in the tumor immune microenvironment (TIME). Co-expression of RNA and protein targets resulted in detection of specific tumor infiltrating immune cell populations and analysis of their activation states.

Conclusions The assay offers a powerful technique for visualizing target RNA biomarkers in specific cell-types identified by cell-marker protein expression. This is a valuable tool for multiomic analysis and accurate interrogation of complex tissues to obtain insights into novel biomarkers and therapeutic targets.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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