title: LNA flow-FISH: a flow cytometry-fluorescence in situ hybridization method to detect messenger RNA using locked nucleic acid probes.
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. beta-Actin mRNA was targeted in whole A549 epithelial cells by hybridization with a biotinylated, LNA-modified probe. The LNA bound to beta-actin was then stained using phycoerythrin-conjugated streptavidin and detected by flow cytometry. Shifts in fluorescence signal intensity between the beta-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 microg/ml of proteinase K for permeabilization and 90 min hybridization at 60 degrees 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.
authors.affiliation: Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, DC 20375, USA. kelly.robertson@nrl.navy.mil
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Robertson
authors.forename: Dzung C
authors.initials: DC
authors.lastname: Thach
mesh_terms: D000199:Actins / Q000235:genetics; D002460:Cell Line; D002463:Cell Membrane Permeability; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005559:Formamides; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000032:analysis* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity; D013696:Temperature; D013995:Time
chemical_list: D000199:Actins; D005559:Formamides; D009841:Oligonucleotides; D012333:RNA, Messenger; C477371:locked nucleic acid; C031066:formamide