Visualizing Gene Expression in Individual Cells in Thick Tissues

STARmap enables simultaneous analysis of multiple RNAs in intact, bulky samples.

Oct 1, 2018
Ruth Williams
SEE-THROUGH: To visualize gene expression in a thick section of brain tissue with various cell types, RNA targets (top) are amplified to create multiple cDNA copies, each containing a barcode specific to the particular gene. Acrylamide is then added to the tissue and cross-linked to the cDNA amplicons (middle). From the resulting hydrogel, fats and proteins are removed to leave a clear tissue within which the barcodes can be sequenced (bottom). See full infographic: WEB | PDF
© george retseck

Cells of a given type or tissue may appear similar and yet behave differently. In the brain, for example, neurons of the same subtype may play very different roles depending on their locations and connections.

In short, when it comes to specific cell functions, “spatial information is absolutely critical,” says gene-expression researcher Je Lee of Cold Spring Harbor Laboratory. Researchers are therefore developing tools to examine the expression of multiple genes in tissue sections, and a technique called STARmap, developed by Xiao Wang, a postdoc in the lab of Karl Deisseroth of Stanford University, and colleagues, is the latest approach.

STARmap (Spatially Resolved Transcript Amplicon Readout Mapping) is “a significant step toward true 3-D gene expression analysis,” says molecular systems biologist Sten Linnarsson of Sweden’s Karolinska Institute who was not involved in the work. In the past, a researcher wanting to examine the expression of multiple genes at once in a tissue was “pretty much limited to working with thin sections” because of imaging difficulties, Linnarsson explains. For example, a principal technique used for such analyses—RNA fluorescence in situ hybridization (FISH)—can suffer from background fluorescence due to probes’ binding to nonspecific sequences and glomming on to tissue proteins.

To maximize the signal-to-noise ratio with STARmap, Wang and her colleagues first amplify their RNA targets within the tissue section using a technique that produces hundreds of tandem cDNA copies, each with a unique DNA barcode. They then add acrylamide to the sample, to which the amplified cDNAs crosslink, forming a tissue hydrogel. Fats and proteins are then stripped away from the gel to leave a transparent yet structurally preserved specimen. Lastly, the team sequences the amplified barcodes in situ, using confocal microscopy to image the patterns of fluorescent nucleotides at individual spots.

“The [image] quality they demonstrate here is very impressive,” Linnarsson says.

The team has used the technique to study the expression of up to 28 genes simultaneously in 150-micron slices of mouse brain tissue, and more than 1,000 genes simultaneously in 8-micron slices. These analyses revealed previously unappreciated differences in the distribution of excitatory and inhibitory neuronal subtypes (identified by their expressed genes) over the brain’s cortical layers.

Next, says Wang, “We are moving up to 1-mm sections, and ultimately aiming for the whole mouse brain.” (Science, doi:10.1126/science.aat5691, 2018)

In-tissue multiplex gene expression detectionTarget RNA
Maximum tissue

Number of expressed genes possible to view at once

Multiplex single-molecule RNA FISHIn situ hybridization of target
mRNAs with fluorescently labeled probes
15 microns

249 in 15-µm slices (Shah et al., Neuron, 92:342–57, 2016)Brain
STARmapIn situ amplification of mRNA targets followed by their in-situ sequencing with ligation of fluorescent probes8 microns for hundreds
of genes

150 microns for tens
of genes
1,020 in 8-µm slices

28 in 150-µm slices