High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in tells and tissues

Oligonucleotides that carry a detectable label can be used to probe for mRN A targets in in situ hybridization experiments. Oligonucleotide probes (OPs ) have several advantages over cDNA probes and riboprobes. These include th e easy synthesis of large quantities of probe, superior penetration of prob e into cells and tissues, and the ability to design gene- or allele-specifi c probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those d irectly conjugated with horseradish peroxidase (HRP). We used branching pho sphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequenc es to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well bot h on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also sh ow that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a-gene family. This work demonstrat es that multihapten-labeled and HRP-conjugated OPs are sensitive and specif ic and can make superior in situ hybridization probes for both research and diagnostic applications.