LNA flow–FISH: A flow cytometry–fluorescence in situ hybridization method to detect messenger RNA using locked nucleic acid probes |
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Authors: | Kelly L. Robertson Dzung C. Thach |
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Affiliation: | aCenter for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, DC 20375, USA;bLaboratory of Infectious Diseases, National Institute of Allergies and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA |
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Abstract: | We present a novel method using flow cytometry–fluorescence in situ hybridization (flow–FISH) to detect specific messenger RNA (mRNA) in suspended cells using locked nucleic acid (LNA)-modified oligonucleotide probes. β-Actin mRNA was targeted in whole A549 epithelial cells by hybridization with a biotinylated, LNA-modified probe. The LNA bound to β-actin was then stained using phycoerythrin-conjugated streptavidin and detected by flow cytometry. Shifts in fluorescence signal intensity between the β-actin LNA probe and a biotinylated, nonspecific control LNA were used to determine optimal conditions for this type of flow–FISH. Multiple conditions for permeabilization and hybridization were tested, and it was found that conditions using 3 μg/ml of proteinase K for permeabilization and 90 min hybridization at 60 °C with buffer containing 50% formamide allow cells containing the LNA-bound mRNA to be detected and differentiated from the control LNA with high confidence (< 14% overlap between curves). This combined method, called LNA flow–FISH, can be used for detection and quantification of other RNA species as well as for telomerase measurement and detection. |
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Keywords: | Fluorescence in situ hybridization (FISH) Flow cytometry Locked nucleic acid (LNA) messenger RNA (mRNA) detection β -Actin |
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