“We now have a ‘Google map’ that allows measuring millions of RNA molecules within the tissue with nanoscale precision, without having to extract them as we did previously,” says Shahar Alon, PhD, of Bar-Ilan University’s Faculty of Engineering, Multidisciplinary Brain Research Center and Institute of Nanotechnology and Advanced Materials. The new technology, Expansion Sequencing or ExSeq, is a significant step forward in efforts to treat complex diseases, such as Alzheimer’s and cancer.

In a research article titled “Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems” published in the journal Science, researchers from Bar-Ilan University, Harvard University, and the Massachusetts Institute of Technology (MIT) reveal that they have succeeded in developing a technology that allows them, for the first time, to pinpoint millions of RNA molecules inside tissues with nanoscale resolution.

Overview of the expansion sequencing technology which enables multiplexed mapping of RNAs at nanoscale resolution throughout intact tissues. Top: schematics of physical expansion and in situ sequencing (left), and image analysis (right). Bottom: characterization of nanoscale transcriptomic compartmentalization in mouse hippocampal neuron dendrites and spines (left, middle), and maps of cell types and states in a metastatic human breast cancer biopsy (right). [Shahar Alon, Daniel Goodwin, Ed Boyden].

“Using expansion technology, researchers and medical doctors will be able to perform genomics analysis in 3D to obtain not only the identity of molecules, but also their location inside the tissue, and thus treat complex diseases better and more effectively,” says Alon, the first author of the study.

ExSeq was created by merging two methods: a method to localize RNA messages within cells (in situ sequencing)  and a method to physically inflate cells to view them better (expansion microscopy). Around six years ago a team at Harvard developed the method of mapping RNA molecules inside cells and another team at MIT developed the method of physically “blowing-up” cells and tissues.

In Alon’s lab researchers are using ExSeq to detect RNA molecules inside nanoscale synapses of neurons in brain tissue. The location of RNA molecules in the brain tissue affects processes such as learning and memory and can shed light on which molecules take part in these processes. This can advance understanding of whether molecules, or their location, are disrupted in certain diseases such as Alzheimer’s.

The new technology can also be used to detect where cancer cells are located in complex tissues in relation to immune system cells, and what RNA molecules they contain. The authors show cancer cells can change their behavior according to the identity of their surrounding cells. For instance, when tumor cells are in the vicinity of immune cells they express a different set of RNA message molecules than when they are far away from immune cells.

Earlier, high-resolution imaging of RNA messages at the nanoscale level has only been possible using sophisticated microscopy techniques that limit the number of RNA molecules that can be viewed simultaneously. On the other hand, methods for highly multiplexed RNA imaging limit the spatial resolution and the ability to localize these RNA messages to subcellular compartments.

This new technology adapts expansion microscopy, which physically expands biological tissues for RNA sequencing with long read-lengths in situ. The authors have applied untargeted-ExSeq to the mouse brain, to identify and localize thousands of genes including genetic variants. Using targeted ExSeq the authors revealed the location of RNA molecules at nanoscale resolution within the tiny spines on the thin dendritic projections of individual neurons in the hippocampus of the mouse brain, a region involved in memory and learning.

The authors show patterns of RNA transcript expression in multiple layers and layer specific cell types in the part of the brain involved in processing visual information. In human metastatic breast cancer biopsy the authors reveal the relative RNA expression patterns of tumor cells in the context of the position of immune cells.

Alon asserts, with this and several other new technologies, the dawn of an age in which it will be possible to create complete molecular maps of tissues from individuals is on the horizon. Experts in the fields of image analysis, data analysis, and genetics will be needed to decipher such huge molecular maps. This radically new approach will offer an unprecedented, detailed understanding of complex diseases as well as normal biological functions in whole organisms.

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