For the first time ever scientists can isolate RNA without damaging surrounding live cells; the technique could help researchers to gain insight into cell function and protein production.
Cells within different types of tissue, such as heart, brain, and skin, are linked by "genes that are transcribed into RNA." This RNA is linked to proteins. Researchers hope to determine how "cell-to-cell chemical connections influence individual cell function and overall protein production," but they need to isolate RNA in order to do that, a Perelman School of Medicine at the University of Pennsylvania news release reported.
Cells that appear to be the same still show variations on the molecular level. The new technique, dubbed TIVA (transcriptome in vivo analysis) could allow researchers to take a closer look.
"Our data showed that the tissue microenvironment shapes the RNA landscape of individual cells," James Eberwine, Ph.D., professor of Pharmacology, Perelman School of Medicine, and co-director of the Penn Genome Frontiers Institute (PGFI), said in the news release.
The non-invasive method allowed researchers to isolate RNA of a single cell within human brain tissue that was taken only minutes after neurosurgery.
The TIVA tag consists of Swiss-Army-Knife-type; it captures mRNA from using "chemical tools." These tools allow the mRNA to be isolated without interfering with neighboring cells. One of these tools is a "sequence of amino acids" that allow the cells to be infiltrated safely
"Rather than targeting a specific cell at this stage, the researchers washed the entire tissue sample from the brain with a solution containing TIVA tags, introducing the molecule to all of the cells," the news release reported.
Since this type of molecule is present throughout the tissue, it is important the "knife" is not activated until it reaches the target cell. A "removable cage" is TIVA's second tool, and it works to keep the tag under control. The "cage" covers the molecule's binding site, preventing it from harvesting the wrong RNA. The signal to bind comes from a laser.
The binding ability comes from an RNA base called uracil, which forms a "poly-U" sequence that binds with a poly-A.
"These molecules are 'caged' in the sense that we're physically blocking their function until we give them the key, which is laser light," Ivan Dmochowski, Ph.D., associate professor of Chemistry, School of Arts and Sciences, co-directed this study said. "Once the cage is off, they're free to do what they want to do: bind to mRNA."
A blue-activating laser allows researchers to break open the cages in a single cell. A green laser is also used to ensure the cage's stability using fluorescent dyes.
"These dyes are positioned in a precise way: one is near the poly-U sequence and the other by the poly-A. When the molecule's cage is still locked, they are close enough together that they can transfer energy from the laser between one another. According to Pharmacology's Sul, probing a cell with a green laser and producing a light emission that corresponds to this energy transfer between the dyes is a signal that the tag has made it into the cell intact," the news release reported.
The team used their new technique to look at gene expressions in isolated neurons and compare them to others in different growing conditions. They found cells in suspension expressed more genes than those residing in intact tissue, "as if the isolated cells were making more RNA to be ready for anything in the absence of getting any meaningful chemical signals from surrounding cells," the news release reported.
The new method could be significant in the future of medical research.
