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.

Clinical implications of detecting low-abundance RNAs by flow cytometry

“…While this technology is a powerful tool for analyzing complex biological events quantitatively and systematically in any cell suspension, its application has been limited primarily to the identification of cell types and functions based on protein expression using specific antibodies, or to cell cycle or ploidy analysis by total DNA staining. Over the last few decades, flow cytometric detection of intracellular RNAs has been attempted by applying various molecular technologies [8,9]. However, the specificity and sensitivity of all previous attempts have not been suitable for the wide range of intracellular RNA analyses, particularly in the case of low-abundance target RNA sequences.…”

Transcriptional profiling of cells sorted by RNA abundance

“…Recently, flow cytometry has been used to sort cells using a spectrum of fluorescent labeling techniques in whicholigonucleotide probes are hybridized to either DNA or RNA target sequences [4][5][6][7] . The principle limitation of these methods has been that RNA extracted from hybridized material is often highly degraded 8,9 .…”

Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry

“…In one study, the investigators employed biotinylated, locked nucleic acid (LNA) probes to detect infection by Sindbis virus (Robertson et al 2010). Although they demonstrated that labeling of the Sindbis genome by in situ hybridization was as effective a method for tracking viral infection as using a recombinant GFP virus, they were unable to study subcellular localization of the viral genome due to the intrinsic limitation of conventional flow cytometry to measure only fluorescence intensity and not localization.…”