Question: Are Tissue Microarrays (TMAs) compatible with the Visium CytAssist Spatial Gene Expression for FFPE assay?
Answer: Tissue Microarrays (TMA) are paraffin blocks that contain cylindrical cores extracted from different tissues. With the Direct Placement (Visium Spatial Gene Expression for FFPE v1) assay, TMAs were unsupported given challenges around sectioning, placement, the risk of core-to-core cross-talk, and complexity of data analysis. With improvements in tissue adhesion, data specificity, and spatiality (see Visium CytAssist Spatial Gene Expression for FFPE: Reagents, Workflow, & Data Comparison Technical Note for assay comparisons), the likelihood of generating quality has improved with the CytAssisted assay. Additionally, with the new analysis tutorial (linked at the end of the article), there’s now guidance for analyzing TMAs with the Visium CytAssist Spatial Gene Expression for FFPE assay. But given that generating TMA blocks, sectioning and tissue placement of TMA sections require expertise, TMAs still pose certain constraints, as noted in the Tested Tissue List.
Outlined below is a summary of our learnings and considerations from limited testing of TMA tissue sections internally. 10x Genomics Support does not have additional information on generating TMA blocks, sectioning, and placing sections beyond what is provided in this article and will not be able to provide additional details from internal experiments or guidance at this time. After tissue slide generation, follow the fully supported 10x Genomics H&E Staining Demonstrated Protocol and User Guide. It is important to note that we have not tested, nor do we support, placement of multiple tissue sections for analysis within a single Capture Area outside of TMAs.
Compatible TMA Features:
- Utilize TMAs with cores originating from the same tissue species as cross reactivity between mouse and human probes can negatively impact assay performance
- TMA configurations tested include 3 x 3 or 5 x 5 arrays, with a minimum of 1 mm pitch between cores (edge to edge measurement; Figure 1)
- Core sizes tested internally range from 1-2 mm diameter
- We have not tested archived TMA tissue slides
- If the tissue array is preserved after hard-set coverslip removal, assess RNA quality of individual punches from another similarly stored archived slide
- If RNA quality is in the recommended range, users can expect TMAs to perform similar to other archived sections
- We have not tested Immunofluorescence (IF) staining with TMA sections, only H&E staining
- Configurations tested internally are listed in Table 1
Figure 1. Example images of sections generated from alternating cores of mouse lung and brain TMAs with supported core size and spacing. 3 x 3 array with 1 mm edge to edge distance between cores (left). 3 x 3 array with 2 mm edge to edge distance between cores (right).
Table 1. Summary of internal TMA experiments.
|TMA Grid||Core Size||Core Distance||Section Thickness||Visium Slide Capture Area|
|Mouse||Brain & Lung*||
|3 x 3||2 mm||2 mm||5 µm||11 mm|
|3 x 3||2 mm||1 mm|
|Brain & Kidney*||
|5 x 5||1 mm||1 mm|
|11 x 6†||1 mm||1 mm|
*indicates cores of alternating tissue types; †indicates only 5 x 5 grid analyzed on Visium Slide.
- We recommend assessing RNA quality of each block prior to generating cores, as described in the Visium CytAssist Spatial Gene Expression for FFPE – Tissue Preparation Guide
- Preparing TMA blocks is very challenging and we recommend using a service provider
- We do not have any guidance on TMA block generation
- We have worked with Acepix Biosciences (Hayward, CA) and Novus Biologicals (Centennial, CO) to generate TMA blocks, in addition to sectioning and placement onto validated glass slides
TMA Sectioning and Placement:
- TMA block sectioning and placement is very challenging and we recommend using a service provider to section TMAs onto blank slides
- Guidance on TMA sectioning and placement is not currently available
- We have tested 5 µm sections from TMA blocks
- We tested compatibility with 11 mm Visium CytAssist Spatial Gene Expression Slides
- 6.5 mm Gene Expression Slides are expected to be compatible but are untested
- Sections larger than the Capture Area dimensions may be used with the assay, but only cores that fit within the Capture Area will have probes captured for downstream library construction
- For example, we placed an 11 x 6 TMA section on a glass slide and performed the 10x workflow on 5 x 5 cores, as those cores would fit within the 11 mm Capture Area (see Table 1)
- After section placement, transfer tissue slides in a slide drying rack to a section dryer and incubate for 3 hours in an oven at 42°C
- Tissue slides can be stored in a room temperature desiccator for up to two weeks before tissue staining
- Subsequently, follow the H&E Staining Demonstrated Protocol before proceeding with the Visium CytAssist Spatial Gene Expression User Guide for library construction
Data Analysis and Datasets:
Products: Visium CytAssist for FFPE