CAMBRIDGE, MA – Using a state-of-the-art device for expanding print, MIT and Harvard Medical School researchers have devised a method to record individual molecules of messenger RNA within a print sample and then classify the RNA.
This approach offers a unique picture of the genes expressed in different parts of a cell, and could allow scientists to learn much more about how cell positioning is affected. on cell location or their interaction with nearby cells. This method could also be useful for mapping cells in the brain or other cigarettes and classifying them according to their function.
“Gene expression is one of the most fundamental processes in all of biology, and it plays roles in all biological processes, both healthy and related to disease. you have more than just having a gene on or off, “he said. Ed Boyden, Y. Eva Tan Professor of Neurotechnology and professor of biological engineering, media arts and sciences, and brain and mental sciences at MIT. “You want to find out where the gene products are. You care what cell types they are in, what individual cells they play in, and even what parts of the cells in which they are. they work. ”
In a study appearing today in Science, the researchers showed that they could use this method to detect and then classify thousands of different messenger RNA molecules inside the mouse brain and in human tumor samples.
The lead authors of the study are Boyden, a researcher at the MIT McGovern Institute and the Howard Hughes Medical Institute; George Church, professor of genetics at Harvard Medical School; and Adam Marblestone, a former MIT research scientist. The paper’s lead authors are Shahar Alon, a former postdoctoral fellow at MIT and now a senior lecturer at Bar-Ilan University; Daniel Goodwin, MIT graduate student; Anubhav Sinha ’14 MNG ’15, MIT graduate student; Asmamaw Wassie ’12, PhD ’19; and Fei Chen PhD ’17, who is an assistant professor of gas and renewable cell biology at Harvard University and a member of the MIT and Harvard Broad Institute.
Knitting extension
The new sequencing strategy builds on a method designed by the Boyden group in 2015 for expanding print samples and then imaging them. By incorporating water-absorbing polymers into a tissue sample, researchers can swallow the tissue sample while keeping the entire body in action. Using this technique, nappies can be enlarged by a factor of 100 or more, allowing scientists to obtain very high resolution images of the brain or other tissues using a regular light microscope.
In 2014, the Church’s laboratory developed an RNA sequencing mechanism called FISSEQ (in situ fluorescent sequence), which allows thousands of mRNA molecules to be detected and sorted within cells grown in a lab basin. . The Boyden and Church laboratories decided to come together to combine tension extension and in situ RNA sequencing, creating a new way of ordering expansion (ExSeq).
Extending the tension before RNA sequencing has two main advantages: It offers a higher view of the RNA in cells, and makes it easier to classify these RNA molecules. “Separating these molecules into the expanding sample, and moving them apart, gives you more room for the in situ ordered chemical reactions. achievement, “Marblestone says.
Once the print is expanded, the researchers can record and classify thousands of RNA molecules in a sample, with a resolution that allows them to identify the locations of the molecules not only in cells but on the other side. within specific regions such as dendrites – the tiny expanses of neurons that receive communication from other neurons.
“We know that the position of RNA in these small areas is important for learning and memory, but until now, we have not had a way to measure these areas because they are very small, in nanometer order,” Alon says.
Using an “untargeted” version of this method, meaning that they do not look for specific RNA sequences, the researchers can convert thousands of different sequences. They estimate that, in a given sample, they can classify between 20 and 50 percent of the genes present.
In the hippocampus of the mouse, this method yielded remarkable results. For one, the researchers found mRNA-containing introns, which are segments of RNA that are normally secreted from mRNA in the nucleus, in dendrites. They also found mRNA molecules encoding transcription factors in the dendrites, which may help with novel forms of dendrite-to-nucleus communication.
“These are just examples of things we would never have looked at on purpose, but now that we can sequence RNA exactly where it is in the neuron, we can do a lot further study of biology, “Goodwin says.
Cell interactions
The researchers also showed that they could study gene expression in a more focused way, looking for a specific set of RNA sequences that correspond to interesting genes. In the visual cortex of the mouse, the researchers used this approach to classify neurons into different types based on a study of 42 different genes that they express.
This technology could also be useful for studying many other types of tumors, such as tumor biopsies. In this paper, the researchers examined breast cancer metastases, which contain many different cell types, including cancer cells and immune cells. The study showed that these cell types can behave differently depending on where they are inside a tumor. For example, the researchers found that B cells that were close to tumor cells expressed some inflammatory genes at a higher level than B cells that were farther from tumor cells.
“The tumor microenvironment has been studied in many different settings for a long time, but it has been difficult to study with any depth,” Sinha says. “A cancer biologist can give you a list of 20 or 30 signaling genes that recognize most cell types in the strain. Here, since we studied 297 different RNA transcripts in the brain. sample, we can ask and answer more detailed questions about gene. emotion. “
The researchers now plan to further study the interactions between cancer cells and immune cells, as well as gene expression in the brain in healthy and diseased states. They also plan to expand their approaches to allow them to map more types of biomolecules, such as proteins, along with RNA.
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The research was funded, in part, by the National Institutes of Health and the National Science Foundation, as well as by Lisa Yang, John Doerr, the Open Philanthropy Project, Cancer Research UK, the Human Atlas Cell Initiative pilot program Zuckerberg, and HHMI.