GeoMx DSP Knowledge Base: Assays & Applications

The UV cleaving area can resolve a single cell. However, 5,000 oligos per target are required for analysis of a given ROI. This translates to 2,500 bound antibodies and 250 RNA molecules per ROI. Therefore, we suggest a minimum of 20 cells in each region of interest (ROI) for protein analysis and 100 cells for RNA analysis. Signal is proportional to collected mass, therefore selecting smaller ROI may result in lack of signal.

Four fluorescent channels are used. Representative dyes include FITC (AF488), Cy3 (AF532), Texas Red (AF594) and Cy5 (AF647).

Yes, it is possible to analyze both protein and RNA expression from individual Area of interest (AOI) on a single slide with up to 18,000 RNA targets and 570+ protein targets across dozens of pathways using GeoMx DSP followed by NGS readout on an Illumina sequencer. For more information on the Spatial Multiomics (formerly Spatial Proteogenomics) workflow, please check out our white paper explaining the entire workflow as well as our manual.

The safest option is to run GeoMx DSP and H&E staining separately, performed using serial sections. If you want to run them on the same slide, then please run the GeoMx DSP workflow first, followed by the H&E staining for histotyping. This strategy has been successful with our internal team for protein slides.

GeoMx DSP works with positively charged slide mounted with 5 µm FFPE tissue sections. It has been validated for fresh frozen as well as fixed frozen samples (Appendices II and III, respectively, in the GeoMx Slide Prep User Manual). Additionally, tissue microarray samples can also be analyzed if the sections are within the recommended scan area (see the green area in the figure below) and at least 2-3 mm apart.

FFPE samples should be fixed with 10% NBF or 4% PFE and embedded in paraffin. We recommend 18-24 hours fixation time for blocks less than 0.5 cm in thickness. The paraffin blocks can be stored long term for future use on GeoMx DSP. Fresh/frozen tissue should be stored in optimal cutting temperature (OCT) media. After sectioning on a cryostat, user can follow the instruction in the manual for either the FFPE or fresh frozen/fixed frozen workflows.

GeoMx DSP is fully validated for samples up to 3 years old prepared from tissue with a cold ischemic time of less than 1 hour using 10% NBF or similar fixative.

Internally, we have tested up to 40-year-old blocks. For 10- to 20-year-old blocks, we have had good signal for RNA. For the 40-year-old block, the signal was low for RNA. Age is not the only consideration here. Cold/warm ischemic time, fixation, storage, etc. all play major roles. For the best results, do not use FFPE blocks that are more than 10 years old.

For GeoMx RNA assays, it is preferable to cut sections within 2 weeks of the time of running samples as oxidation of the RNA molecules begins shortly after cutting the section. Cut sections should be stored at 4° C with a desiccant pack. RNA quality is important for GeoMx DSP experiment with the RNA panel. RNA may be degraded in old sections, especially the low expressors. If concerned about the RNA quality, in situ hybridization or RNAscope can be performed on these samples and if there is good signal for low or middle expressors, then samples are okay to use for GeoMx DSP experiment. Please see the direct link to ACD’s control probe options. The most common kit for qualifying samples prior to GeoMx DSP is the RNAscope 3-plex positive control kit that matches the species (please see human here).

Protein is less sensitive to age, preservation, and storage conditions than RNA and customers may find a higher success rate for protein analysis on older blocks.

Bruker Spatial Biology recommends using positively charged slides. Internally, we have had the most success using Superfrost plus or Leica BOND plus slides (Catalog No. S21.2113.A). Although not validated by Bruker Spatial Biology, products like Epredia™ Tissue Section Adhesive (Fisher Scientific, Catalog No. 86014) or TOMO® Adhesion Microscope Slides (VWR, Catalog No. 10000-038) may also work well.

Citrate buffer and TRIS-EDTA both work with RNA assay; however, the number of targets below the LOQ is high when using citrate buffer for RNA assay. This is one of the steps that was extensively tested by our R&D team, hence our recommendation has always been to use TRIS-EDTA and not deviate from it for RNA work.

For our assays, the critical requirement for the water is that it is nuclease-free and RNase-free. Both DEPC-treated and nuclease-free water sources should meet these criteria and can be used in the GeoMx DSP workflow. (While DEPC can impact downstream enzymatic activity, most commercial DEPC-treated water is tested for inactivity post-treatment). Please note that the Spatial Multiomics (previously Spatial Proteogenomics) assay workflow calls specifically for nuclease-free water in the preparation of 1X TBS-T, not DEPC-treated water.

For RNA assays, the target retrieval conditions, and the Proteinase K digestion conditions vary among tissues. Our internal team has tested some tissues and shared the conditions that worked, highlighted below and printed in the GeoMx DSP Manual Slide Prep User manual. Target retrieval and Proteinase K conditions were determined based on FFPE tissue blocks meeting the constraints outlined in the sample guidance section of the GeoMx DSP Manual Slide Prep User manual. Samples were primarily tumor, with minimal normal adjacent tissue. These conditions may vary by sample, the amount of normal adjacent tissue, and other factors. These conditions were optimized for large tumor sections and may not apply to arrayed tissues, cored tissues, and needle biopsies.
Please note that if your tissue of interest is not mentioned here, we recommend using default conditions for the target retrieval (15 min at 100°C) and Proteinase K digestion (1 ug/mL at 37°C) steps.

Morphology markers are used by researchers to reveal tissue structures which are relevant to the research questions by helping to select regions of interest (ROI) and if desired, ROI segmentation into cell-type compartments. Directly conjugated primary fluorescent antibodies is the most straight forward approach. Bruker Spatial Biology offers several morphology marker kits for the commonly used markers. In addition, our internal teams have compiled a list of markers that have been used successfully internally, which can be accessed here. If conjugated antibodies are not an option, one can choose alternative approaches such as using antibody conjugation kits, secondary antibody staining protocol, as well as RNAscope for RNA assay. For more information on these approaches, please refer to our tech note on morphology markers here.

GeoMx nuclear staining kit includes a DNA dye (SYTO13) which is detected in the FITC/525 nm channel. You may consider using a different SYTO DNA dye (SYTO 83 and SYTO Far Red; sold by Thermo Fisher Scientific) to free up the FITC or Cy3 channels for other markers, if required. We do not recommend using DAPI for DNA staining for GeoMx DSP experiments. This is because UV light, which is required for DAPI excitation, is reserved for barcode cleavage.

For our Morphology Kits, the antibodies in the protein-compatible and the RNA-compatible kit are the same (exception: human CD45 antibody is different between RNA and protein kits) but in a different concentration. The antibodies in the RNA-compatible kit are 2X the concentration of those in the protein-compatible kit, so that the final concentration on the tissue is 2X compared to the protein-compatible antibodies. This is to compensate for the differences in slide prep methodology in the protein and RNA workflows. Therefore, you can use the Protein-compatible morphology markers with your RNA samples, please just use twice the starting amount of antibody when preparing your solutions for the slides.

Bruker Spatial Biology has observed the best tissue adherence with Leica BOND Plus slides and they are the preferred slide type for tissues with known poor adherence. In addition to this, extending the baking time to 37°C overnight followed by 65°C 3-4 hours may also help. For both the CosMx SMI and GeoMx DSP workflows, adjustments to the high-temperature antigen or target retrieval step may help preserve tissue adhesion. Fat-rich tissues, such as breast cancer, may benefit from shorter incubation times to avoid complete loss of adipose tissue. Delicate tissues and cell pellets may benefit from lowering target retrieval temperatures. Consult the Slide Preparation User Manual or the above FAQ on Target Retrieval and Proteinase K conditions.

The Spatial Multiomics (formerly Spatial Proteogenomics) assay could be performed manually instead of semi-automated BOND RX/RX workflow. The standard RNA slide preparation protocol in the GeoMx DSP Manual Slide Preparation user manual should be followed up to the Proteinase K digestion step, however, please use 0.1 µg/mL concentration instead of 1 µg/mL concentration. After the digestion step, the rest of the workflow can be followed as in the Spatial Proteogenomic Assay User Manual. (This workflow may also be referred to as Spatial Multiomics). Please note that this is not validated by Bruker Spatial Biology, thus some empirical testing may be needed on your end.

For GeoMx DSP assays with nCounter readout, you can spike in 5-10 targets of your choice.

For GeoMx DSP assays with NGS readout, protein panels such as GeoMx IO Proteome Atlas can accommodate spike in of up to 40 targets. For RNA assays with NGS readout, you can spike in up to 400 targets, which would have to be split into two 200-target spike-in add-on panels. In addition, customers can also design a standalone panel of only 200 targets with an additional 200 targets as add-on.

• WTA + 200 add-on + 2nd 200 add-on
• Standalone 200 + 200 add-on

Unfortunately, due to the complex nature of miRNA, we do not currently have a method to detect microRNA on GeoMx DSP.

We can design probes to detect circRNAs; however, to specifically detect a circRNA, we need to design the probe to straddle the back splice junction, and as a result, we can only get one probe for each circRNA. Therefore, there may be some sensitivity issues with these targets. In addition, since we have not validated circRNA detection, we would recommend including robust controls to assess the specificity of the probes.

Storage and shelf life for the GeoMx DSP reagents can be found in the table below. Storage conditions for GeoMx DSP reagents are listed on the individual reagent container as well as in the GeoMx DSP Slide Preparation user manual. These conditions include room temperature, 4˚C, -20˚C and -80˚C. Please read labels carefully to ensure correct storage and product integrity.

All GeoMx DSP reagents have a shelf-life of at least 1 year from date of manufacture, and for most items, the shelf life is 2-3 years from date of manufacture. Bruker Spatial Biology guarantees that products will have at least 3 months’ shelf-life remaining upon shipment.

To determine the expiration date of a reagent, please refer to the reagent packaging, which will show the date of expiration or the date of manufacture. The date of expiration is indicated by the hourglass symbol

and indicates the use-by date. The date of manufacture is indicated by the manufacturer symbol

and indicates the date the product was manufactured; refer to the table below for the shelf-life of the reagent from the data of manufacture.

Seq Code and Pro Code plates should be stored at -20°C. Once thawed, the plates can be stored up to 3 months at 4° C. Alternatively, the plates may be re-frozen and can go through up to 3 freeze-thaw cycles.

Safety Data Sheets (SDSs) can be found through the Resources page on our website or directly from the Bruker Spatial Biology home page under Support and Resources > Product Support > Safety Data Sheets. You may choose GeoMx DSP under instrument category to find the SDS specific to this instrument.