Comparison of 3' and 5' biotin labelled oligonucleotides for in situ hybridisation

Oligonucleotide probes enzymatically labelled at the 3'-end with biotin have been used successfully to detect target RNA and DNA in combination with in situ hybridisation. Addition of multiple biotin residues to the 3'-end increases the hybridisation signals, but it is not known whether the same principle is applicable to the 5'-end. We have labelled a 35-base oligonucleotide during synthesis with 1, 5 and 12 biotin molecules at the 5'-end and compared it to conventional 3'-labelling. In additional experiments the probes were labelled at both ends. Probes were applied to histological sections obtained from paraffin-embedded cell-clot-complexes that contain uninfected and Rhinoviral-infected cells, using a standard in situ hybridisation protocol with appropriate controls. Hybridisation signals were compared for intensity of cytoplasmic signal and sensitivity as number of positive cells. Both parameters increased in parallel with higher numbers of biotin residues attached to the 5'-end and 12 biotin residues were almost as effective as 3'-enzymatic tailing. The sensitivity could be increased above that of either 3'- or 5'-labelling by the addition of residues at both ends of the probe. The 5'-attachment of biotin residues can extend the value of oligonucleotide probes employed for in situ hybridisation and yield increased sensitivity when combined with 3'-enzymatic labelling.