Scientists demonstrated—for the first time—that the combination of locked nucleic acid (LNA) probes with a flow cytometry-fluorescence in situ hybridization (LNA-flow-FISH) method can be used to quantify viral RNA in infected cells. The new method allows scientists to detect the presence of viruses in cells and to study their growth, as well as monitor the changes in viral RNA accompanying antiviral drug treatment.
Scientists at the Naval Research Laboratory (NRL) Center for Bio-Molecular Science and Engineering, working in tandem with researchers at the NRL Lab for Biosensors and Biomaterials (both in Washington, DC), were responsible for demonstrating the LNA-flow-FISH method with the LNA probes. Once the LNA probe is bound to the viral RNA inside mammalian cells, it is tagged with a fluorescent dye, and then thousands of these tagged cells are measured rapidly by flow cytometry.
The LNA probe differs from traditional nucleotide probes by binding more tightly to its target RNA. While nucleotide probes are highly sensitive, they involve the lysis of cells prior to measurement, making them unable to provide information about cellular viability, infected cell phenotypes, percentage of infected cells or the variation in infection among a cell population.
LNA-flow-FISH can screen for compounds with antiviral activity quickly and could be adapted for monitoring infections in the blood for vaccine therapy and development. This method adds a necessary tool for several emerging areas in cell biology that enables the use of high throughput measurements for entire populations and improves statistical analyses.
"This method can be expanded by adding more than one kind of LNA probe to enable multiple detection of different viral and host RNA," says Kelly Robertson, a postdoc at NRL. "The multiplexing enhancement can be used to better understand infectious agents, allowing this technique to be used to aid in the development of antiviral drugs for a variety of viruses."
One exception to the LNA-flow-FISH method is viral strains that have not acquired genetic mutations, known as wild-type viruses (such as strains of HIV), which would require a large initial investment of labor for engineering each virus of interest.
Posted by Lee Mather
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