Abstract B095: Multiplexed detection of RNA and proteins on the nCounter® platform with low sample input protocol
Authors: Warren Sarah , Geiss Gary , Burditt Brian , Mei Qian , Huang Alan , Eagen Maribeth , Ignacio Eduardo , Dunaway Dwayne , Dennis Lucas , Beechem Joseph 
Published: November 2016

Abstract
As our understanding of the immune responses to cancer continues to grow, the need to extract greater amounts of information from ever smaller sample sizes increases. One of the biggest challenges facing the field is to develop a comprehensive understanding of how the immune system responds to a tumor, and multi-omic profiling (DNA, RNA, and protein) is crucial to building a holistic model of tumor immunity. The NanoString nCounter platform has become an important tool in quantifying transcriptional responses from a wide variety of sample types by enabling direct digital counting of up to 800 targets from a single sample using nucleic acid probes that directly hybridize to the RNA sequence of interest and then are quantified with optical barcoding technology. The platform has now been extended to permit quantification of proteins at the same time using primary antibodies conjugated to DNA oligos that hybridize with the barcodes. We have developed new technology that enables characterization of up to 30 proteins and 770 RNA transcripts in key immuno-oncology pathways from a very low amount of starting material - as few as 50,000 cells. NanoString has previously developed the nCounter Vantage RNA:Protein Immune Profiling Panel which allows digital counting of extracellular proteins which facilitates quantitation of multiple immune cell populations and provides information about their activation status. We have now applied this technology to detect intracellular and secreted proteins as well in the new nCounter Vantage RNA:Protein Immune Signaling panel. This panel is able to detect key transcription factors, signaling molecules, and secreted proteins from peripheral blood mononuclear cells (PBMC), dissociated cells, or cell culture. Additionally, we have recently developed a universal cell capture technology that utilizes anti-β2M antibody coupled to magnetic beads to pull down nucleated cells from a large starting volume. As proof of concept, PBMC from a healthy donor were treated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, TNFα, or IFNγ and RNA and protein were characterized with both the Immune Profiling and Immune Signaling panels. nCounter protein detection compared favorably when validated with flow cytometry, and the additional information imparted by simultaneous profiling of the RNA transcriptome enabled mapping of signaling pathways activated by treatment. Furthermore, RNA and protein counts from the low input protocol were representative of counts obtained from higher cell inputs, indicating that no loss or skewing of data resulted from reducing the starting material. This advance in multi-analyte, multiplexed digital molecular profiling will accelerate immuno-oncology research by reducing sample size requirements and may enable the discovery and development of novel immunotherapies and their associated companion diagnostics.
Citation Format: Sarah Warren, Gary Geiss, Brian Burditt, Qian Mei, Alan Huang, Maribeth Eagen, Eduardo Ignacio, Dwayne Dunaway, Lucas Dennis, Joseph Beechem. Multiplexed detection of RNA and proteins on the nCounter® platform with low sample input protocol [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B095.