Transglutaminase-Mediated In Situ Hybridization (TransISH) for mRNA Detection in Mammalian Tissues

Abstract

In the most commonly used in situ hybridization (ISH) procedure, a hapten-labeled antisense nucleic acid (e.g., RNA) probe is employed to hybridize a target mRNA in tissue sections. The hapten-labeled RNA is then detected by a highly specifi c hapten–antibody interaction; however, it requires laborious immunostaining steps for spatial coloring on tissue sections. To simplify ISH-based mRNA detection systems, we created a new RNA–(enzyme) n conjugate for sensitive detection of mRNA in tissue sections. In the present simple, antibody-free ISH protocol, an antisense RNA probe of interest is fi rst modifi ed with a specifi c dipeptide substrate of microbial transglutaminase (MTG) by in vitro transcription. Alkaline phosphatase from a hyperthermophile is then covalently linked to the substrate-labeled RNA by MTG-catalyzed sitespecifi c conjugation. A robust, multi-enzyme-labeled RNA probe enables the direct labeling of a target mRNA in tissue sections with signaling enzymes under harsh hybridization conditions, leading to one-step signal amplifi cation after hybridization. The application of the new trans glutaminase-mediated ISH (TransISH) strategy to mRNA detection in mammalian tissues was demonstrated.