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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).

Table 1: Standard enzymatic procedures for the preparation of non-radioactive RNA/cRNA probes.

TemplateMethodLabeled ProbeLabeling SiteEnzyme
DNA in vitro Transcription cRNA random T7- / SP6- / T3-RNA Polymerase
RNA 3’ End Labeling RNA 3’ OH Terminal deoxynucleotidyl Transferase (TdT)
RNA 3’ OH Yeast Poly A Polymerase
RNA 3’ OH T4 RNA Ligase
5’ End Labeling RNA 5’ OH T4 Polynucleotide Kinase (T4 PNK)