3'-end fluorochromized and haptenized oligonucleotides as in situ hybridization probes for multiple, simultaneous RNA detection

We have used fluorescein-, digoxigenin- and biotin-(di)deoxyXTPs and terminal deoxynucleotidyl transferase for small scale labeling of synthetic oligonucleotide probes and here we show the applicability of such probes for the in situ detection of multiple RNA sequences. The enzymatic 3'-end-labeling methods proved to be good alternatives for the chemical fluorochrome and hapten labeling of 5'-end alkylamino-derivatized oligonucleotides. By combining 3'-end fluorescein-, biotin-, and digoxigenin-labeled oligonucleotides, double and triple hybridizations are feasible. For example, we demonstrated simultaneously mRNAs coding for caudodorsal cell hormone, a molluscan insulin-related peptide, and 28 S ribosomal RNA in cryostat sections of the cerebral ganglia of the pond snail Lymnaea stagnalis.