nanoString GeoMx Digital Spatial Profiler

 

General Features

NanoString’s GeoMx Digital Spatial Profiler (DSP) combines spatial and molecular profiling technologies by generating digital whole transcriptome or protein expression data for up to 4 tissue slides per day. A series of imaging reagents tagged with fluorophore are used to visualize the tissue and a series of oligo-tagged profiling reagents are used to interrogate protein or transcript expression. Once regions of interest (ROIs) are selected by the investigator, DNA oligos bound to the profiling reagents are sequentially released, collected into individual wells on a 96-well plate, and subjected to Illumina library prep. Expressions levels are readout using an Illumina Sequencer and analyzed using the DSP interactive software.

 

 

Supported Assays

  • Whole Transcriptome Atlas (human or mouse)
  • Cancer Transcriptome Atals (human)
  • Protein Assays with NGS readout (human or mouse)

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Sample Requirements

  • FFPE or Fresh/Frozen tissue
  • Species Specific – only human and mouse samples currently supported
  • Recommended section thickness is 5 µm
  • Tissue should be sectioned onto positively charged slides no more than 2 weeks prior to your Spatial Assay Appointment
  • A test slide for a scanning only run (without ROI definition, UV illumination, or ROI collection) is strongly recommended to expedite ROI selection the day of the experiment and validate the fluorescent staining of your morphology markers. All requests utilizing custom antibodies are required to undergo this process. 

 

Reagents

AGC GeoMx DSP Reagents June 2021 – present

Vendor Part Number Description
nanoString  GeoMx NGS RNA CTA Hs GeoMx Cancer Transcriptome Atlas
nanoString GeoMx NGS RNA WTA Hs GeoMx Human Whole Transcriptome Atlas
nanoString GeoMx NGS RNA WTA Mm GeoMx Mouse Whole Transcriptome Atlas
nanoString various Protein Module for NGS

 

DSP Workflow

The nanoString GeoMx DSP platform is highly interactive and projects require extensive planning. Schedule a consultation here. Once your assay has been selected, additional key decision points are:

Slides and Samples: Multiple sections or sections from multiple tissues can be placed on the same slide for DSP processing. The only caveat is that all sections must fit within the GeoMx gasket. The gasket dimensions are shown here.  

Recommended slides:

  • Leica Bond Plus (Cat# S21.2113.A)
  • Fisherbrand Superfrost Plus (Cat # 12-550-15) 

Morphology Marker Selection: Morphology markers are employed to guide selection of ROIs based on visual staining patterns. While we stock nanoString morphology kits that contain common tissue markers, the DSP system is agnostic to the imaging reagents. We have a list of antibodies previously validated by the nanoString Technology Access Program. Custom antibodies are also supported as long as the antibody conjugate works with one of the 4 channels on the instrument. One of the fluorescent channels needs to be reserved for a nuclear stain, leaving three channels open for antibodies of interest. The list of available fluorescent channels are:

Channel Excitation Peak/Bandwidth Emission Peak/Bandwidth
FITC / 525 nm 480 / 28 nm 516 / 23 nm
Cy3 / 568 nm 538 / 19 nm 564 / 15 nm
Texas Red / 615 nm 588 / 19 nm 623 / 30 nm
Cy5 / 666 nm 645 / 19 nm 683 / 30 nm

Defining Regions of Interest: There are multiple approaches for defining ROIs, including placing geometric shapes around specific areas (Geometric), drawing concentric circles surrounding a histological landmark (Contour), and gridding across the entire slide (Gridded). 

Image provided by nanoString

You can select 1-96 ROIs per slide as long as they do not overlap with each other. ROI size determines total counts collected. The largest allowable ROI is ~700 μm. While the smallest region for illumination is 10 μm, this is insufficient for generating profiling data. Ultimately, the minimum ROI size will depend on your tissue, the types of cells within your ROIs, and, for RNA, the transcriptional activity of the cells. We do not recommend starting with fewer than 50 cells per ROI (non-segmented). If segmenting, then larger ROIs will be required.

Regions can also be profiled using immunofluorescence/RNAscope patterns (segmentation, cell type specific). These are defined by combinations of the presence and absence of specific morphology markers will become areas of illumination (AOIs) during slide processing. Each AOI gets collected separately, thereby creating two or more expression profiles for the same ROI.

Considerations when creating segment masks include: 

  • Segments cannot overlap or extend outside an ROI 
  • Segments should be as contiguous as possible, with clean margins separating each segment from off-target tissue
  • Segments composed of numerous small particles will have reduced fidelity and higher background from adjacent tissue

Defining ROIs and their segmentation pattern is a lengthy process and requires a significant time commitment on the investigators part the day of the experiment. All first time users are required to come to NCRC for ROI selection.

Data Analysis: The AGC will complete the nanoString NGS pipeline to generate the digital count (DCC) files and notify you when they have been loaded onto the DSP. To perform data analysis, please reserve time on the instrument using our nanoString GeoMx Data Analysis calendar in MiCores. Please note days/times the instrument is available for analysis is limited based on projects the AGC is processing on the device

 

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