RNA and cRNA fragments of variable length ("probes") are able to form non-covalent highly specific duplexes with a complementary nucleic acid strand (termed hybridization). When a label is attached to such a hybridization probe it can thus efficiently serve for detection of a defined DNA or RNA target sequence. Labeled RNA/cRNA probes are routinely used e.g. for (fluorescence) in situ hybridization ((F)ISH), micro-array based gene expression profiling or electrophoretic mobility shift assay (EMSA) experiments.
The most commonly used labels for the generation of non-radioactive RNA/cRNA probes are fluorophores and haptens, the latter meaning Biotin, Desthiobiotin, Digoxigenin and Dinitrophenol. Fluorophores are directly detected by fluorescence spectroscopy whereas the visualization of haptens requires secondary reporter molecules (= indirect labels).
Fluorophores and haptens are enzymatically introduced into RNA/cRNA via modified nucleotides that are incorporated as substitutes for their natural counterparts in either a one-step procedure (introduction of a fluorescent or hapten-modified nucleotide) or a two-step procedure (introduction of a reactive group carrying nucleotide and subsequent fluorescent or hapten labeling).