Monitoring viral RNA in infected cells with LNA flow-FISH

  1. Eddie L. Chang1
  1. 1Laboratory for Biosensors and Biomaterials, Code 6910, Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, District of Columbia 20375, USA
  2. 2Department of Molecular and Microbiology, George Mason University, Manassas, Virginia 20110, USA
  3. 3Laboratory of Infectious Diseases, Department of Health and Human Services, National Institute of Allergies and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Abstract

We previously showed the feasibility of using locked nucleic acid (LNA) for flow cytometric–fluorescence in situ hybridization (LNA flow-FISH) detection of a target cellular mRNA. Here we demonstrate how the method can be used to monitor viral RNA in infected cells. We compared the results of the LNA flow-FISH with other methods of quantifying virus replication, including the use of an enhanced green fluorescent protein (EGFP) viral construct and quantitative reverse-transcription polymerase chain reaction. We found that an LNA probe complementary to Sindbis virus RNA is able to track the increase in viral RNA over time in early infection. In addition, this method is comparable to the EGFP construct in sensitivity, with both peaking around 3 h and at the same level of infected cells. Finally, we observed that the LNA flow-FISH method responds to the decrease in levels of viral RNA caused by antiviral medication. This technique represents a straightforward way to monitor viral infection in cells and is easily applicable to any virus.

Keywords

Footnotes

  • Reprint requests to: Eddie L. Chang, Laboratory for Biosensors and Biomaterials, Code 6910, Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375, USA; e-mail: eddie.chang{at}nrl.navy.mil; fax: (202) 767-9511.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2016410.

  • Received November 24, 2009.
  • Accepted May 20, 2010.

Freely available online through the RNA Open Access option.

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  1. Published in Advance June 28, 2010, doi: 10.1261/rna.2016410 RNA 16: 1679-1685

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