Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization

Detection of target RNA by in situ hybridization (ISH) in the classic and c onfocal fluorescence microscope was performed using strand-specific single- stranded RNA probes labeled directly with the fluorochromes fluorescein iso thiocyanate or Texas red. The probes, produced by in vitro transcription fr om PCR-generated templates with T7 RNA polymerase and fluorochromized UTP, gave ISH signals directly visible by fluorescence microscopy without the us e of any immunological detection step. In avoiding antibodies, it was possi ble to strongly increase the sensitivity of the TSH since antibodies may co ntain RNase which can reduce hybridization signals considerably, even beyon d the detection limit. Fluorescent RNA probes thus allowed for the detectio n of low numbers of target molecules per cell, such as minus strand interme diates in picornavirus RNA replication. Using appropriate denaturing condit ions, the targets could be visualized in a double-stranded configuration as well as in the presence of a 100-fold excess of complementary RNA. Further more, double ISH for the simultaneous detection of two different RNA specie s, such as plus and minus strand RNA of poliovirus, or of different regions of the viral genomic RNA was possible with appropriate fluorescent strand- specific probes labeled with different fluorochromes. Combination of ISH an d immunofluorescence was found feasible if RNA was present in relatively la rge amounts. In addition to the investigation of virus replication, possibl e applications of fluorochromized RNA probes might include antisense RNA de tection as well as plant virus resistance and gene silencing.