title: Monitoring viral RNA in infected cells with LNA flow-FISH.
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.
authors.affiliation: Laboratory for Biosensors and Biomaterials, Code 6910, Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Robertson
authors.forename: Anne Brooks
authors.initials: AB
authors.lastname: Verhoeven
authors.forename: Dzung C
authors.initials: DC
authors.lastname: Thach
authors.forename: Eddie L
authors.initials: EL
authors.lastname: Chang
mesh_terms: D000818:Animals; D000998:Antiviral Agents; D002460:Cell Line; D002477:Cells / Q000737:chemistry; D006224:Cricetinae; D005434:Flow Cytometry / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000235:genetics / Q000379:methods*; D009696:Nucleic Acids / Q000235:genetics; D055585:Physical Phenomena; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic; D014779:Virus Replication / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D009696:Nucleic Acids; D012333:RNA, Messenger; D012367:RNA, Viral; D012313:RNA