Spatial transcriptomics is a method for assigning cell types (identified by the mRNA readouts) to their locations in the histological sections and can also be used to determine subcellular localization of mRNA molecules. First described in 2016 by Ståhl et al., it has since undergone a variety of improvements and modifications.
The Ståhl method implies positioning individual tissue samples on the arrays of spatially barcoded reverse transcription primers able to capture mRNA with the tails. Besides tail and spatial barcode, which indicates the x and y position on the arrayed slide, the probe contains a cleavage site, amplification and sequencing handle, and unique molecular identifier. Commonly, histological samples are cut using cryotome, then fixed, stained, and put on the microarrays. After that, it undergoes enzymatic permeabilization, so that molecules can diffuse down to the slide, with further mRNA release and binding to the probes. Reverse transcription is then carried out in situ. As a result, spatially marked complementary DNA is synthesized, providing information about gene expression in the exact location of the sample. Thus, described protocol combines paralleled sequencing and staining of the same sample. It is important to mention that the first generation of the arrayed slides comprised about 1,000 spots of the 100-μm diameter, limiting resolution to ~10-40 cells per spot.
In the broader meaning of this term, spatial transcriptomics includes methods that can be divided into five principal approaches to resolving spatial distribution of transcripts. They are microdissection techniques, Fluorescent in situ hybridization methods, in situ sequencing, in situ capture protocols and in silico approaches.
Application
Defining the spatial distribution of mRNA molecules allows for the experimentalist to uncover cellular heterogeneity in tissues, tumours, immune cells as well as determine the subcellular distribution of transcripts in various conditions. This information provides a unique opportunity to decipher both the cellular and subcellular architecture in both tissues and individual cells. These methodologies provide crucial insights in the fields of embryology, oncology, immunology and histology. The functioning of the individual cells in multicellular organisms can only be completely explained in the context of identifying their exact location in the body. Spatial transcriptomics techniques sought to elucidate cells’ properties this way. Below, we look into the methods that connect gene expression to the spatial organization of cells.
Microdissection
Laser capture microdissection
Laser capture microdissection enables capturing single cells without causing morphologic alterations. It exploits transparent ethylene vinyl acetate film apposed to the histological section and a low-power infrared laser beam. Once such beam is directed at the cells of interest, film directly above the targeted area temporarily melts so that its long-chain polymers cover and tightly capture the cells. Then, the section is removed and cells of interest remain embedded in the film. This method allows further RNA transcript profiling and cDNA library generation of the retrieved cells.
RNA sequencing of individual cryosections
RNA sequencing of the selected regions in individual cryosections is another method that can produce location-based genome-wide expression data. This method is carried out without laser capture microdissection. It was first used to determine genome-wide spatial patterns of gene expression in cryo-sliced Drosophila embryos. Essentially, it implies simple preparation of the library from the selected regions of the sample. This method had difficulties in obtaining high-quality RNA-seq libraries from every section due to the material loss as a result of the small amount of total RNA in each slice. This problem was resolved by adding RNA of a distantly related Drosophila species to each tube after initial RNA extraction.