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A team of researchers from the University of Illinois at Urbana-Champaign and the Mayo Clinic has developed a new type of molecular probe capable of measuring and counting RNA in cells and fabrics without organic dyes. The probe is based on the conventional in situ fluorescence hybridization (FISH) technique, but relies on compact quantum dots to illuminate diseased molecules and cells rather than fluorescent dyes.
Over the last 50 years, FISH has evolved into a multi-billion dollar industry because it can efficiently represent and count DNA and RNA in unique cells. However, FISH has its limitations because of the delicate nature of the dyes. For example, dyes deteriorate rapidly and are not very good in three-dimensional imaging. In addition, conventional FISH can only read a few RNA or DNA sequences at a time.
"By replacing dyes with quantum dots, there is no stability problem and we can count many RNAs with higher fidelity than before," said Andrew Smith, an associate professor of biochemistry. engineering and member of the research team. "In addition, we discovered a fundamental limit to the size of a molecular marker in cells, revealing new design rules for analysis in cells."
In their latest article, published on October 26, 2018, in the online edition of Nature CommunicationsSmith and his team have identified an optimal size for quantum dots to work effectively with the FISH protocol. This discovery allowed FISH based on quantum dots to match the labeling specifications currently obtained with organic dyes.
The team created unique quantum dots made of zinc alloy, selenium, cadmium and mercury and coated with polymers. "The core of the dot dictates the emission wavelength and the hull, the amount of light emitted," said Smith, also affiliated with the Micro + Nanotechnology Lab, at Carle Illinois College of Medicine and at the Department of Materials Science. and engineering at the University of Illinois.
These points can emit colors regardless 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 the number of dye-based FISH cells decreased rapidly in minutes. The FISH-based quantum dot method provided a long-term luminescence allowing the counting of RNA for more than 10 minutes, allowing the acquisition of 3D cell imaging.
"This is important because cell and tissue images are acquired step by step, so subsequent sections marked with dyes are depleted before they can be replicated," Smith said.
This research is part of the Mayo Illinois Alliance in which Illinois engineers work directly with clinicians and biologists from the Mayo Clinic to solve outstanding medical problems. The Mayo Clinic's biomarker discovery group is working on the development of FISH-based diagnostics for tumor biopsies to improve the accuracy of cancer diagnosis, select personalized treatments, and improve outcomes. prognoses. The QD-FISH methodology was developed to address this need for biomarker panels requiring the analysis of numerous genetic changes in tumor cells for which only small amounts of specimens may be available when they are acquired. with a thin needle, as is often the case in prostate cancer. .
This research was funded by the Mayo-Illinois Alliance, the National Institutes of Health and the National Science Foundation. The full title of the article is "Improved mRNA FISH with compact quantum dots".
In addition to Smith, the following Illinois bioengineering researchers contributed to the work: Yang Liu, Phuong Le, Sung Jun Lim, Liang Ma and Suresh Sarkar. Mayo Clinic collaborators include Dr. Farhad Kosari, Stephen Murphy, John Cheville and George Vasmatzis.
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