LNA flow-FISH: a flow cytometry-fluorescence in situ hybridization method to detect messenger RNA using locked nucleic acid probes.

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.