Researchers from the University of Illinois at Urbana-Champaign bioengineering team and Mayo Clinic have engineered a new type of molecular probe that can measure and count RNA in cells and tissue without organic dyes. The probe is based on the conventional fluorescence in situ hybridization (FISH) technique, but it relies on compact quantum dots to illuminate molecules and diseased cells rather than fluorescent dyes. This research is published in Nature Communications.
|Quantum dots illuminate the locations of individual mRNA as red dots in the cytoplasm of a single HeLa cell. The blue region is the nucleus. Credit: University of Illinois at Urbana-Champaign Department of Bioengineering|
Over the last 50 years, fluorescence in situ hybridization technique has evolved into a multi-billion-dollar industry because it effectively images and counts DNA and RNA in single cells. However, fluorescence in situ hybridization technique has its limitations due to the delicate nature of the dyes. For example, the dyes rapidly deteriorate and are not very good at imaging in three dimensions. In addition, conventional fluorescence in situ hybridization technique can only read out a couple of RNA or DNA sequences at a time. Using quantum dots, however, can illuminate the locations of individual mRNA as red dots in the cytoplasm of a single HeLa cell.
The team created unique quantum dots that are made of a zinc, selenium, cadmium, and mercury alloy and are coated with polymers. “The core of the dot dictates the wavelength of emission, and the shell dictates how much light will be given off,” said Smith, who is also affiliated with the Micro + Nanotechnology Lab, Carle Illinois College of Medicine, and Department of Materials Science and Engineering at the University of Illinois.
These dots can emit color independent of the size of the particle, which is not the case for conventional quantum dots. The dots are also small enough (7 nanometers) to fit on a probe that can maneuver between proteins and DNA in a cell, making them more comparable in size to the dyes used in conventional FISH probes.
In experiments with HeLa cells and prostate cancer cells, the researchers found that dye-based FISH cell counts declined rapidly in minutes. The quantum dot-based FISH method provided long-term luminescence to allow counting of RNA for more than 10 minutes, making it possible to acquire 3D cell imaging.
Citation: Liu, Yang, Phuong Le, Sung Jun Lim, Liang Ma, Suresh Sarkar, Zhiyuan Han, Stephen J. Murphy, Farhad Kosari, George Vasmatzis, John C. Cheville, and Andrew M. Smith. “Enhanced mRNA FISH with Compact Quantum Dots.” Nature Communications 9, no. 1 (2018). doi:10.1038/s41467-018-06740-x.