A Novel Amplification Method of Nonradioactive In Situ Hybridization Signal for Specific RNA with Biotinylated Tyramine

For a better performance of nonradioactive in situ hybridization for specific RNA sequences, signal amplification is sometimes required especially in the use of clinical specimens processed under suboptimal conditions and in the analysis of gene expression with very low reiteration. In the present study, we examined the usefulness of catalyzed signal amplification (CSA) or catalyzed reporter deposition system with biotinylated tyramine to amplify colorimetric in situ hybridization signals. Our CSA protocol included the use of biotinylated tyramine after the reaction of thymine-thymine (T-T) dimerized DNA with horseradish peroxidase (HRP) labeled anti-(T-T dimer) antibody, followed by the reaction with HRP-labeled streptavidin. When thymine-thymine dimerized lambda phage DNA was spotted onto a nitrocellulose filter at 1 pg-10 ng and then detected by the direct immunoperoxidase method, the indirect immunoperoxidase method or the CSA method, the CSA method gave the highest sensitivity with about 100-fold Increase compared to that of the direct method. Next, in the frozen sections of rat brain, which was fixed with 4% paraformaldehyde, 28S rRNA staining by in situ hybridization with only 10 ng/ml of T-T dimerized oligonucleotide probe complementary to rat 28S rRNA was compared among the direct method, the HRP-labeled avidin-biotin complex method and the CSA method. Again, intense 28S rRNA signal was obtained only with the CSA method. Therefore, we confirmed the usefulness of the catalyzed deposition system with biotinylated tyramine in nonradioactive colorimetric in situ hybridization for specific RNA sequences in tissue sections.