Fluorescent In Situ Sequencing Helps Diagnose Sick Tissues Early

Fluorescent in situ sequencing has potential applications, such as new diagnostics that spot the earliest signs of disease.

Fluorescent in situ RNA sequencing (FISSEQ), could lead to earlier cancer diagnosis by revealing molecular changes that drive cancer in seemingly healthy tissue. It can track cancer mutations and their response to modern targeted therapies, and uncover targets for safer and more effective ones. The method could also help biologists understand how tissues change subtly during embryonic development––and even help map the maze of neurons. The researchers described the method in the February 2014 online edition of Science.

The Wyss Institute of Biologically Inspired Engineering at Harvard University and Harvard Medical School (Boston, MA, USA) in collaboration with the Allen Institute for Brain Science (Seattle, WA, USA) developed the new method that allows scientists to pinpoint thousands of mRNAs and other types of RNAs in intact cells, while determining the sequence of letters. Messenger RNAs (mRNAs) are positioned throughout living tissues, and their location often helps regulate how cells and tissues grow and develop. Until today, in order to analyze many mRNAs simultaneously, scientists have had to grind cells to a pulp, which left them without a method to pinpoint their location within the cell.

“By looking comprehensively at gene expression within cells, we can now spot numerous important differences in complex tissues like the brain that are invisible today,” said George Church, a professor of genetics at Harvard Medical School and a core faculty member at the Wyss Institute. “This will help us understand like never before how tissues develop and function in health and disease.”

Healthy human cells typically turn on nearly half of their 20,000 genes at any given time, and they choose those genes carefully to produce the desired cellular responses. Moreover, cells can dial gene expression up or down, adjusting to produce anywhere from a few working copies of a gene to several thousand. Prof. George Church and Je Hyuk Lee, a research fellow at Harvard Medical School and the Wyss Institute wanted to pinpoint the cellular location of the mRNAs. They wanted to simultaneously determine the sequence of those RNAs, which identifies them and often reveals their function.

The team first treated the tissue chemically to fix the cell’s thousands of RNAs in place. Then they used enzymes to copy those RNAs into DNA replicas, and copy those replicas many times to create a tiny ball of replica DNA fixed to the same spot. They managed to fix and replicate thousands of the cell’s RNAs at once––but then the RNAs were so tightly packed inside the cell that even a tricked-out microscope and camera could not distinguish the flashing lights of one individual ball of replica DNA from those of its neighbors.

To solve that problem the researchers assigned the cell a unique address: the sequence of “letters,” or bases, in the RNA molecule itself. They figured they could read the address using methods akin to next-generation DNA sequencing, a set of high-speed genome sequencing methods Prof. Church helped develop in the early 2000s.

The scientists sought to fix RNA in place in the cell, make a tiny ball with many matching DNA replicas of the RNAs, then adapt next-gen DNA sequencing so it worked in fixed cells. The four flashing colors would reveal the base sequence of each replica DNA, which would tell them the base sequence of the matching RNA from which it was derived. And those sequences would in theory provide an unlimited number of unique addresses––one for each of the original RNAs.

The method, called fluorescent in situ RNA sequencing (FISSEQ), could lead to earlier cancer diagnosis by revealing molecular changes that drive cancer in seemingly healthy tissue. It could track cancer mutations and how they respond to modern targeted therapies, and uncover targets for safer and more effective ones.

Related Links:
Allen Institute for Brain Science
Wyss Institute of Biologically Inspired Engineering at Harvard University and Harvard Medical School