Transglutaminase-Mediated in Situ Hybridization (TransISH) System: A New Methodology for Simplified mRNA Detection
2012
Kitaoka, Momoko | Mitsumori, Masayuki | Hayashi, Kounosuke | Hiraishi, Yoshiyuki | Yoshinaga, Hisao | Nakano, Koji | Miyawaki, Katsuyuki | Noji, Sumihare | Goto, Masahiro | Kamiya, Noriho
Detection and localization of specific DNA or RNA sequences in cells and tissues are of great importance for biological research, diagnosis, and environmental monitoring. However, the most common procedure for in situ hybridization employs laborious immunostaining techniques. In the present study, we report proof-of-concept for a new RNA-enzyme conjugated probe for the detection of mRNA on tissue sections with a simple procedure. An RNA probe modified with a specific dipeptide substrate of transglutaminase was prepared. Alkaline phosphatase was then covalently and site-specifically combined to the dipeptide-labeled RNA using microbial transglutaminase. The new RNA probe labeled with alkaline phosphatase was validated by in situ hybridization (ISH) and proved to be a sensitive and sequence specific probe for mRNA detection in tissues. The new transglutaminase-mediated ISH (TransISH) strategy is free from antigen–antibody reaction, leads to one-step signal amplification after hybridization, and thus will be widely applicable for highly sensitive nucleic acid detection.
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