Cells don’t operate in isolation. They constitute and reside in highly complex, interconnected tissue systems. They exert biological influence on their surroundings and are functionally differentiated by signals they receive from neighboring cells and tissues.
To understand a tumor’s response to a drug, researchers must study more than the tumor itself. They must look at the microenvironment, the interplay between cancer and stroma, and the influence of the immune system. The Visium Spatial Gene Expression Solution incorporates unbiased, high-throughput total mRNA analysis for intact tissue sections with morphological context, offering a much needed multidimensional view of tissue biology. Visium also provides an easy way to get started with spatial transcriptomics, only requiring access to a cryosection, a microscope, and a sequencer, bridging genomics and tissue pathology. Some tips and recommendations for scientists who are just getting started to better understand aspects that are unique to the Visium workflow are highlighted here.
Fresh-frozen tissue is first sectioned and placed onto a slide with thousands of barcoded spots, each containing millions of capture oligonucleotides with spatial barcodes unique to that spot. Standard fixation and staining techniques, including hematoxylin and eosin (H&E) staining, are utilized, and tissue sections are visualized on the slides. The tissue is then permeabilized, releasing mRNA, which binds to capture oligos, from a proximal location on the tissue. A reverse transcription reaction occurs while the tissue is still in place, generating a cDNA library that can be mapped back to specific spots within the tissue (Figure 1).
Tips for sample preparation
To maintain RNA quality and tissue morphology, the tissue must be frozen rapidly. One method, snap freezing, entails preparing tissue in an isopentane and liquid nitrogen bath. It is best to avoid freezing the tissue directly in liquid nitrogen, as this could cause unpredictable cracking of the tissue. Alternatively, the tissue can be frozen and embedded simultaneously in optimal cutting temperature compound (OCT). Visium is currently
compatible with fresh-frozen tissue; however, 10x Genomics scientists are exploring use cases with paraformaldehyde and formalin-fixed paraffin-embedded (FFPE) tissues as well.
To successfully section tissue for the slides, consider utilizing the following tips:
- Prior to sectioning the tissue, it may be necessary to make adjustments to the sample temperature and orientation.
- To avoid damaging tissue morphology, layer a small amount of OCT on top of the tissue and allow it to refreeze.
- It is critical to place tissue sections within the fiducial borders of the square capture areas. Fiducials orient the section spatially and enable the Space Ranger data analysis pipeline to properly detect barcoded spots within the tissue. Once the tissue section is placed, adhere the tissue by putting your finger on the back of the slide, behind each section. Replace the slide within the cold plate of the cryostat to refreeze the tissue.
Tissue samples are precious and often difficult to acquire. To get the most robust data from a sample, it is essential to fine-tune the experimental workflow to match optimal permeabilization conditions. Tissue optimization (TO) for the spatial gene expression experiment should be performed with representative tissue sections. Ideally, these will be nearly identical to the target sections in both anatomical region and thickness. TO slides then undergo an optimization protocol where they are permeabilized for different lengths of time to assess the release of mRNA—registered as a fluorescence signal—for each timepoint. This process is particularly important when comparing healthy controls and diseased tissue, which may have different cellular compositions and optimal
Visium utilizes gold-standard methods for H&E tissue staining to color the nuclei, cytoplasm, and connective tissue of the cells within your tissue sections. This solution has been optimized for the Nikon Eclipse Ti2 with brightfield and fluorescence capacity; however, it is compatible with any equivalent imaging system that can export images as TIFF or JPEG files. It is important to ensure that no water droplets remain on the slides after the stain is completed, as droplets can interfere with imaging the tissue, distorting the light from the microscope as it passes through the water.
From tissue to gene expression data
Permeabilization is essential to release mRNA from the tissue. This reaction occurs on top of the slide in a slide cassette that seals around the capture areas. When you load the Visium spatial gene expression slide into the cassette, ensure the slide is face down, so that the tissue is exposed to reaction solutions after the cassette is flipped over. Additionally, label the outside of the cassette as the original slide label will be covered once the slide is loaded.
Finally, after you load the permeabilization enzyme into the wells of the cassette, tap the device gently against your hand or lab bench to ensure the enzyme covers the whole capture area. This will ensure that all areas of the tissue release mRNA.
The final output of all wet lab steps is a sequencing-ready cDNA library. After the cDNA is sequenced, data is analyzed and visualized with 10x Genomics’ software tools, Space Ranger and Loupe Browser, to yield a high-resolution microscope image of the tissue section with overlaid gene expression data (Figure 2).
The Visium will allow scientists to bring multidimensional depth to their research. With access to the transcriptome of an entire tissue section, they are no longer limited to exploring single targets or areas using traditional single-modal analysis tools. They can now discover gene expression at high resolution, while simultaneously interrogating cellular behavior within the tissue microenvironment.