nCounter Knowledge Base: Assays & Applications

Understanding the role of tissue/disease type is crucial when planning a CosMx SMI experiment. Tissue biology, as well as tissue block and section quality, should be considered with each new experiment.
For the RNA Assay, we recommend starting with default conditions as per the CosMx Slide Preparation Manual. For some tissues, digestion buffer concentration and/or incubation time for target retrieval or digestion may require adjusting and will need to be empirically determined. For example, if tissue detachment is observed during slide preparation, stop sample preparation, and begin again with a new tissue section where a reduced target retrieval time is used. If the issue persists, consider reducing the Proteinase K concentration. For tissue-specific experimental condition examples see the Top 3 Tips for a Successful CosMx SMI Run. These conditions have been used by NanoString’s Technology Access Program (TAP). Note: not all conditions used by TAP have been validated. Researchers should always begin with default experimental conditions before testing any of these modifications. Slide processing conditions should be tested in advance to guarantee tissue adherence. Researchers may choose to use an antibody of choice to visualize tissue structure integrity.

No, a performance difference is not expected because 10% (v/v) neutral buffered formalin (NBF) and fresh 4% (w/v) formaldehyde solution are both made from paraformaldehyde powder and contain almost identical concentrations of formaldehyde (3.7% vs 4% respectively). In NBF, formaldehyde concentration is given as a percent volume per final volume (v/v), whereas in PFA it is given as a percent weight per final volume (w/v).

10% NBF often contains methanol as an additive, which results in 10% NBF penetrating tissue faster than 4% PFA. Therefore, there may be small differences in the time required for tissue fixation between 10% NBF and 4% PFA. Be sure to follow the CosMx SMI slide preparation protocol and adhere to the same conditions for all samples in a particular study.

The length of fixation has a direct influence on the preservation of RNA over time and on the methods required to make the RNA accessible for probe binding. It thus influences the RNA counts obtained in a CosMx RNA assay. Our recommendation is to use a “standard” fixation time of 18-24 hours; however, the optimal fixation time may vary depending on tissue type. Consider your own knowledge of your samples and their performance in similar assays e.g. RNAScope™.

Performance in an RNAScope assay (such as the RNAScope 3-plex positive control kit) is the best measure for probe accessibility to RNA in the tissue. Other sample quality testing, such as DV₂₀₀ values, will not be as informative since they measure the breakdown of the RNA. Fragmentation is not the problem with long fixation – the issue is accessibility of the RNA within the tissue while keeping the morphology intact.  (As described in a subsequent answer, DV₂₀₀ is a useful proxy for the general quality of the sample.)

If fixation time is too short the RNA in the tissue will degrade over time and the digestion steps during sample prep might need to be scaled down to accommodate the more fragile tissue. Failing to scale down the digestion steps may result in reduced RNA counts.

If fixation time is too long, the tissue can be overcross-linked, and the RNA may not be as accessible for probe binding. This would manifest as low counts in the RNA assay. To optimize, both target retrieval and protease digestion steps should be prolonged to counteract the longer fixation. The right conditions need to be empirically determined, and extreme fixation may not be recoverable by these protocol modifications.

Additionally, there are fixation methods that should not be used for CosMx samples. For example, formic acid should not be used as it can degrade nucleic acids, especially with longer fixation times, which will reduce the signal in the CosMx SMI RNA assay.

In protein assays, over-fixation can mask epitope binding sites, reducing antibody probe binding to targets and reducing counts in the assay.

Metrics such as DV₂₀₀ or RIN ranges may be used to determine tissue section quality (if both options are available, DV₂₀₀ is more accurate).  DV₂₀₀ is a metric primarily used in RNA sequencing (RNA-seq) applications. It measures the percentage of RNA fragments in a sample that are longer than 200 nucleotides (nt) in length. Higher DV₂₀₀ values (e.g., DV₂₀₀ > 90%) indicate a higher proportion of intact RNA molecules and are generally considered a good indicator of RNA sample quality. It suggests that a significant portion of the RNA is intact and suitable for sequencing. Lower DV₂₀₀ values (e.g., DV₂₀₀ < 70%) indicate that the RNA sample may be degraded to some extent.

RIN values range from 1 to 10, with higher values indicating better RNA integrity and lower values suggesting RNA degradation. Samples with RIN values above a certain threshold are typically considered to have great RNA signal (e.g., RIN > 7).

Higher RIN scores and DV200 values may predict a stronger RNA signal; however, because the CosMx SMI system detects RNA targets with probes that bind 50-60 nt in the RNA, samples with lower scores are also viable for analysis on the CosMx SMI platform.

Alternatively, if the tissue has been previously run on the GeoMx DSP platform, you can use the GeoMx DSP data to select high-quality RNA samples that are suitable for CosMx SMI experiments. If you plan to run both GeoMx DSP and CosMx SMI experiments, it’s advisable to begin with the GeoMx DSP project. This way, you can identify which RNA samples perform well and subsequently utilize them for your CosMx SMI experiments.

Performing an RNAScope assay is the best measure for sample quality being suitable for a CosMx experiment, if not running a GeoMx experiment on the same samples. With an RNAscope positive control probe suitable samples will have greater than or equal to 4-9 dots per cell with none or very few of the dots in clusters (≥2 score based on the ACD score guidance).

For more information on sample quality, see the second installment of Tips When Generating Single-Cell Spatial Data: Sample Selection available now at NanoString.com.

Tissue sections must be centered within the Scan Area (the green area) of the slide and be no larger than 20 mm long by 15 mm wide (see image below; not to scale). For best performance, ensure that some tissue-free glass is present in all four corners and within the center of the scan area (that is, within the dashed teal line). For examples of tissue placement best practices, see Appendix I in the CosMx SMI Slide Preparation User Manuals available in NanoString University.

It is also important to note the tissue orientation on the slide and how that orientation is then visualized on the instrument and in AtoMx. This graphic is available in the CosMx SMI Instrument User Manual and may help guide you with tissue placement and orientation.

NanoString has put together a Sample Sectioning Tips and Tricks document that guides you through FFPE block preparation and sectioning for use on the CosMx SMI. This document is available now at NanoString University.

Additionally, please see our Tips When Generating Single-Cell Spatial Data post on the NanoString website that is focused on tissue and sample preparation. This post covers not only tissue sectioning but other valuable sample relevant tips such as block quality, and section quality.

Yes! Custom panels are available for both RNA and Protein assays through the CosMx SMI Custom Solutions page. The CosMx RNA Barcoding Service (RBS) enables you to create customized RNA content for use with CosMx SMI. Swap in 7-50 targets of your choice to our pre-validated 1000-plex RNA assay (7-10 targets for our 100-plex RNA assay) or add up to 200 custom targets to the CosMx 6K Human Discovery Panel. You can also build your own entirely custom CosMx SMI RNA panel for up to 350 targets for spatial analysis of any species. Download the CosMx Custom RBS order form and work with your local Sales representative to build your custom panel.

CosMx SMI protein panels can be customized through the CosMx Custom Protein Barcoding Service to barcode up to 8 additional antibodies of interest for use with the CosMx 64-plex Protein Assay (to a maximum of 72-plex). Make sure to choose targets that are abundantly expressed to achieve a strong and specific signal (refer to the white paper Selection and Validation of CosMx Custom-Labeled Antibodies). In addition, choose an antibody that has been recommended or has been validated for immunofluorescent (IF)/ immunohistochemistry (IHC) assays by the antibody provider, preferably in the same antigen retrieval buffer as used in CosMx Protein assays (citrate buffer, pH 6.0). Antibodies must be provided in carrier-free buffer and free of BSA, gelatin or cell culture supernatant. While antibodies in glycerol-containing buffers can still be used for conjugation, overall conjugation efficiency may be affected. Addition of sodium azide is recommended to avoid potential contamination during storage. Work with your local Sales representative or Field Application Scientist for more information.

The first step to take once you’ve received your custom panel/add-on is to email AtoMxKitAdmin@nanostring.com at least one business day prior to a planned run to have the custom kit added into your CosMx SMI Control Center. This step must be performed to ensure that the custom panel/add-on will appear in the dropdown menu during flow cell record creation.

During slide preparation, use the tables found in the Slide Preparation User Manual (for RNA or Protein) to determine the correct volume of custom panel or add-on to use during the hybridization or incubation step. For the Protein assay, this volume may vary depending on the final concentration of the custom antibodies.

Prior to loading the instrument, ensure that the correct imaging tray is being used. The CosMx SMI Imaging Tray is analyte-, plex-, and slide-number-dependent. Custom panels of up to 100 total targets use the 100-plex Imaging Tray; larger custom panels (up to 350 targets total) require the use the 1000-plex Imaging Tray. (It’s recommended to check with the NanoString Bioinformatics team to confirm the correct run specifications for your custom panel.) Check the tray label before use to ensure the correct imaging tray has been selected. If running a Protein Assay and using custom barcoded antibodies, you will need to add custom reporters to the tray during the “Load Instrument” step of the instrument workflow. The tray well to be used is printed on the custom antibody tube. Make sure to note the correct well before instrument loading. Reference the CosMx SMI Instrument User Manual for imaging tray and bulk reagent options.

When setting up the run on-instrument, once at the Flow Cell Configuration screen, complete the Reagent Configuration field by selecting the correct Core Probe Panel and Add-ons (where applicable). If the correct custom add-on or panel is not listed, contact NanoString Support at support@nanostring.com. If running a Custom Protein assay, a custom add-on panel confirmation screen will pop up once the custom add-on panel has been selected. Ensure the selected panel matches the instructions in the pop-up; however, do not add the reporters until immediately before loading the tray into the instrument.

If running a custom RNA assay, continue with flow cell configuration and instrument loading as normal. For custom protein assays, after flow cell configuration is complete, load the custom reporters into the Imaging Tray. Custom reporters are added to well A10, B10 or both depending on the number of custom antibodies being added. Each custom antibody stock vial will indicate which reporter it is associated with. Once the custom reporters have been added, continue with the workflow as normal.

No, segmentation marker customization is not yet available for CosMx SMI assays. CosMx SMI segmentation markers are labeled with a CosMx-specific readout domain, rather than directly conjugated to a fluorophore, so commercial kits provided by antibody vendors cannot be used to custom-label antibodies for CosMx SMI.

Gene lists can be found by visiting the product page for the assay of interest: CosMx RNA Assay or CosMx Protein Assay. On the product page select the panel you are interested in and scroll to the bottom of the panel page. Gene lists are available in the Support Documents or Related Resources section. You must complete a form that includes your email address before the gene list download is available.

To request a new product or product feature, submit an email to support@nanostring.com or use the Contact Us form on the NanoString website.

It is recommended to always use the reagents listed in the CosMx slide preparation protocol as these have been validated. To date, few reagent substitutions have been tested for CosMx slide preparation aside from the ones listed below.

Citrisolv/Xylene

It has been determined that Citrisolv may be used in lieu of Xylene during the Deparaffinization step of the RNA workflow (Day 1 Slide Prep). This substitution requires extending the Ethanol wash step following the Citrisolv wash to 5 minutes. For the Protein assay, although not yet tested, our internal R&D team does not anticipate any issues with using Xylene in lieu of Citrisolv, with no other modifications to the protocol needed. These modifications are listed in the CosMx SMI Slide Preparation User Manuals available in NanoString University.

Deionized Formamide

If 100% deionized formamide is not available, formamide that is not deionized may be used. However, it has been determined that deionized is optimal for the assay’s performance.

At this time, only Leica BOND Plus slides and VWR Superfrost Plus Micro Slide Premium have been validated for use on the CosMx and we do not recommend using different glass slides. NanoString has observed the best tissue adherence with Leica BOND Plus slides and they are the preferred slide type for tissues with known poor adherence.

TOMO® Adhesion Microscope Slides, Matsunami Glass (VWR, 10748-166) have also shown suitable tissue adherence in preliminary testing. Although they have not yet been fully validated, they may serve as a suitable alternative.

Storage conditions for CosMx SMI reagents are listed on the individual reagent container as well as in the CosMx SMI Slide Preparation Manuals. These conditions include room temperature, 4˚C, -20˚C and -80˚C. Please read labels carefully to ensure correct storage and product integrity.

All CosMx 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. Nanostring 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.

Safety Data Sheets (SDSs) can be found through the NanoString Resources page or directly from the NanoString home page under Support and Resources > Product Support > Safety Data Sheets.

Waste should be disposed of according to your facility’s waste disposal procedures. To properly follow your facilities guidelines, read and understand the Safety Data Sheets (SDSs) for all reagents being used (available on the NanoString Support and Resources page). While most reagents provided by NanoString are not considered hazardous, the imaging tray reagents and liquid waste from the waste container contain Proclin and must be disposed of in a manner suitable to your facility.

Some customer-provided reagents, such as ethanol, xylene, and formamide are also considered hazardous and should be stored and disposed of according to your facility’s procedures.

Always be sure to read, understand and maintain SDSs for all chemicals and reagents according to your facility’s requirements.