SENSITIVE MESSENGER-RNA DETECTION BY FLUORESCENCE IN-SITU HYBRIDIZATION USING HORSERADISH PEROXIDASE-LABELED OLIGODEOXYNUCLEOTIDES AND TYRAMIDE SIGNAL AMPLIFICATION

With the ongoing progress in human genome projects, many genes are dis covered whose function and/or expression pattern are not known. Most o f these genes are expressed in relatively low abundance compared to ho usekeeping genes such as elongation factor-la and p-actin. Gene expres sion is studied by Northern blot assays or by semiquantitative PCR met hods. Another method is the visualization of transcripts in tissue or cell cultures by fluorescence in situ hybridization (FISH). However, f or low-abundance RNA detection, this method is hampered by its limited detection sensitivity and by the interference of background signals w ith specific hybridization signals. Background signals are introduced by nonspecific hybridization of probe sequences or nonspecific binding of antibodies used for visualization. To eliminate background signals derived from both sources and to benefit from the peroxidase-driven t yramide signal amplification (TSA), we directly conjugated horseradish peroxidase (HRP) to oligodeoxynucleotides (ODNs) and used these probe s to study in the bladder cancer cell line 5637 the expression of vari ous cytokine genes which, according to Northern hybridization and reve rse transcriptase-polymerase chain reaction (RT-PCR) assays, are expre ssed at levels up to 10,000-fold less than abundantly expressed housek eeping genes. The results show that reduction of probe complexity and the limited use of immunocytochemical detection layers strongly reduce s noise signals derived from nonspecific binding of nucleic acid probe and antibodies. The use of the HRP-ODNs in combination with TSA allow ed detection of low-abundance cytokine mRNAs by FISH.