MGC Spatial Gene Expression

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The relationship between cells and their organization within tissue is critical to understanding the context in which cells function in their microenvironment. The advent of spatial expression profiling is facilitating the characterization of cell-cell and cell neighborhoods during critical phases of development and disease. The MGC is pleased to offer two spatial profiling solutions that can profile RNA and proteins at near- or single cell resolution.

Spatial profiling of RNA: The 10xGenomics Visium Spatial Gene Expression Solution incorporates unbiased, high-throughput total mRNA analysis for intact tissue sections with morphological context, offering a previously inaccessible view of tissue biology. Preserving spatial context while identifying distinct groups of cells offers critical information to understand the relationship of cellular function, phenotype, and location in tissue microenvironments.

Visium fresh frozen: Fresh-frozen tissue samples are sectioned and placed in either two or four capture areas on the Visium Spatial Gene Expression slide. Using standard fixation and Hematoxylin and Eosin (H&E) staining  tissue sections are first visualized on slides. The tissue is then permeabilized to release mRNA from the cells with mRNA binding with spatially-barcoded oligonucleotides present on the spots. A reverse transcription reaction produces cDNA from captured mRNA. The second strand of cDNA is then synthesized and denatured. The barcoded cDNA is then pooled for downstream processing and library preparation to complete a sequencing-ready library. The Visium Spatial Gene Expression library is sequenced using standard short-read sequencers, and data is processed and visualized using 10x Genomics software: Space Ranger Analysis Pipelines and Loupe Browser, and third party applications.

 

Spatial Gene Expression

 

MGC Visium Fixed Tissue

Visium fixed tissue: The 10xGenomics Visium Spatial Gene Expression for FFPE solution has been specifically designed to accommodate fragmented target RNA that occurs during the fixing and embedding process. The spatial FFPE solution uses a panel of 18,000 paired 25mer probes that are ligated on the target RNA and contain an oligo sequence that is captured by the poly-A primers on the surface of the Visium slide (as above). This new capability will enable investigators to profile archival banked FFPE samples to spatially resolve RNA expression in human or mouse FFPE tissue.

Spatial profiling of proteins: While spatial profiling of RNA within cells provides an incredible snapshot into a cells activity at the time of tissue excision, cannon in the field dictates that changes in RNA expression do not always equate to change in protein expression. Further, the field of molecular biology has relied on canonical protein markers that do not always equate to characteristic RNA markers. To address these gaps in our understanding the MGC is pleased to announce the implementation of the Akoya Phenocyler.

Akoya Phenocycler: Currently available to a series of stakeholders within the Duke School of Medicine, the Phenocycler allows for profiling of panels of up to 100 proteins at true single cell resolution. The protocol uses a one-time staining of tissue with barcoded antibodies and cyclical hybridization of fluorescent reporter molecules whose images are captured across the tissue using our Zeiss microscope. Data are analyzed using third party software to carryout differential expression, cell-cell, and cell-neighborhood analyses. The Akoya platform will be available all Duke Researchers in early 2023.

Akoya Phenocycler