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The blood proteome holds great promise for precision medicine but poses daunting challenges due to the low abundance of the majority of plasma proteins and the vast dynamic range across the proteome. We report the development and validation of a novel proteomic analysis technology – NUcleic acid Linked Immuno-Sandwich Assay (NULISA™) – that incorporates a dual capture and release mechanism to suppress the assay background to the minimum, thus drastically improving the signal-to-noise ratio. NULISA improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level, which is enabled by antibody-conjugated DNA sequences that mediate the purification of immunocomplexes and contain target-and sample-specific barcodes for next-generation sequencing-based, highly multiplexed analysis. To demonstrate its performance and utility, we developed a 200-plex NULISA targeting 124 cytokines and chemokines and 80 other immune response-related proteins that demonstrated superior sensitivity for detecting low-abundance proteins and high concordance with other immunoassays. The ultra-high sensitivity enabled the detection of previously difficult-to-detect but biologically important, low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA uniquely addresses longstanding challenges in the proteomic analysis of liquid biopsy samples and makes broad and in-depth proteomic analysis accessible to the general research community and future diagnostic applications.

Complex and highly heterogenous tissues such as the brain are comprised of multiple cell types and states with exquisite spatial organization. Single cell RNA sequencing (scRNA-seq) is now being widely used as a universal tool for characterizing known and novel cell populations within these heterogenous tissues, ushering in a new era of single cell biology. However, the use of scRNA-seq presents some limitations due to the use of dissociated cells, which results in the loss of spatial organization. Incorporating a multiplexed spatial approach that can interrogate gene expression with single cell resolution in the tissue context is a powerful addition to the scRNA-seq workflow. In this study, we used the RNAscope Multiplex Fluorescent and RNAscope HiPlex in situ hybridization (ISH) assays to confirm and spatially map the diverse striatal neurons that have been previously identified by scRNA-seq in the mouse brain (Gokce et al, Cell Rep, 16(4):1126-1137, 2016). We confirmed the gene signatures of two discrete D1 and D2 subtypes of medium spiny neurons (MSN). The heterogenous MSN subpopulations were marked by a transcriptional gradient, which we could spatially resolve with RNA ISH. Numerous striatal non-neuronal cell populations were also identified by scRNA-seq. Finally, the spatial relationship between the D1 and D2 MSN subtypes identified by Gokce et al. was visualized using the RNAscope HiPlex assay, which allows for detection of up to 12 RNA targets simultaneously in intact tissues. In conclusion, we have demonstrated the utility of two multiplexed RNAscope ISH assays for the confirmation and spatial mapping of scRNA-seq transcriptomic results, of the heterogenous mouse striatum at the single cell level. Incorporating spatial mapping by the RNAscope technology into single cell workflows complements scRNA-seq results and provides additional insights into the cellular organization and functional states of diverse cell types in healthy and disease tissues.

The tumor microenvironment (TME) is a network of complex interactions between the tumor and surrounding immune cells. To identify new predictive biomarkers to better stratify patients, it is essential to comprehensively characterize the immune cells within the TME at the molecular level. We have developed a novel workflow combining the single molecule and single cell visualization capabilities of the RNAscope in situ hybridization (ISH) assay with the highly multiplexed spatial profiling capabilities of the GeoMx(TM) Digital Spatial Profiler (DSP) RNA assays (Research Use Only). The fully automated RNAscope Multiplex Fluorescent assay was used to visually identify CD3E (T-cell)-enriched regions and CD19 and CD20 (B-cell)-enriched regions within FFPE human lung cancer tissues. Using the GeoMx DSP, 10 CD3E-enriched regions of interest (ROI) and 10 CD19-enriched ROI were spatially profiled for 78 genes related to immune-oncology research. The RNAscope Multiplex Fluorescence assay was used again to visually confirm the differentially expressed genes between the T and B-cell-enriched regions with single cell resolution. To show a workflow combining RNAscope molecularly guided visualization and GeoMx DSP profiling is feasible, we confirmed that both assay protocols are compatible and tested the fully automated workflow. We compared the differentially expressed genes within the T cell and B cell-enriched ROI. The RNAscope assay confirmed that the CD3E (T-cell)-enriched ROI demonstrated significantly higher expression of the checkpoint markers (PD-1, PD-L1, CTLA4, ICOSLG) as well as inflammatory markers (CCL5, CXCL9, IFNG) compared to the CD19-enriched ROI. We present a robust workflow that overcomes the historical limitations of ISH and IHC by combining high resolution imaging with high plex profiling. With this workflow, the RNAscope ISH technology can molecularly guide the GeoMx DSP to precisely profile ROI while retaining the morphological context.

Gao-Fei Ren,1 Li-Li Xiao,2 Xiao-Jun Ma,1 Yu-Shan Yan,1 Peng-Fei Jiao3 1Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, People’s Republic of China; 2Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, People’s Republic of China; 3Department of Respiratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, People’s Republic of ChinaCorrespondence: Peng-Fei JiaoDepartment of Respiratory, The First Affiliated Hospital of Zhengzhou University, No. 1, Jianshe East Road, Zhengzhou 450052, Henan Province, People’s Republic of ChinaTel +86 371-66913257Email 314761380@qq.comPurpose: Patients with type 1 diabetes (T1D) are associated with a high risk of multiple complications, so the development of T1D treatment is urgently needed. This study was set out to explore the molecular mechanism of metformin in the treatment of T1D insulin resistance.Patients and Methods: Subcutaneous adipose tissues were collected from 68 T1D patients and 51 healthy controls. Insulin resistance model rats and cells were constructed and treated with metformin respectively. Western blot was used to detect p53 and RAP2A protein levels, and qPCR was utilized to measure p53 and RAP2A mRNA levels. SiRNA and RAP2A siRNA vectors were constructed to observe their effects on insulin resistance model cells.Results: In T1D, p53 was up-regulated, while RAP2A was down-regulated. Metformin could effectively improve insulin resistance and inflammatory response while down-regulating p53 and up-regulating RAP2A. P53 induced insulin resistance and inflammatory response by inhibiting RAP2A and promoted apoptosis.Conclusion: Metformin improves T1D insulin resistance and inflammatory response through p53/RAP2A pathway, and the regulation of p53/RAP2A pathway is conducive to improving the efficacy of metformin in the treatment of insulin resistance.Keywords: type 1 diabetes, p53, RAP2A, insulin resistance, metformin