A METHOD FOR PREVENTING ARTIFACTUAL BINDING OF CRNA PROBES TO NEURONSCAUSED BY IN-SITU HYBRIDIZATION

When in situ hybridization was used for the detection of mRNA for the beta-trace protein (beta-trace; prostaglandin-D-synthase) in sections of rat and porcine brains, unspecific binding reactions of sense and a ntisense probes to neurons were observed. The p-trace fragment which s erved as a template for the synthesis of cRNA probes was blunt end-clo ned in the vector pCR-Script SK (+). It was demonstrated that the unsp ecific signals were caused by artifactual binding of two portions of t he cRNA which correspond to sequences of the multicloning site of this vector. These sequences are localized between the SrfI restriction si te (or the insert) and the promoter for the T7 RNA polymerase. Thus, a rtifactual binding could be prevented using riboprobes synthesized by T3 RNA polymerase instead of T7 RNA polymerase. Because of the relativ ely weak transcription efficiency of T3 RNA polymerase, as compared wi th T7 RNA polymerase, a blocking procedure was established which allow ed successful in situ hybridization with T7 RNA polymerase-synthesized probes. Blocking was performed using synthetic oligonucleotides deduc ed from the two sequences of the multicloning site which were found to be responsible for artifactual binding. (C) 1998 Academic Press.