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 specific 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 first modified with a specific 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 site-specific 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 amplification after hybridization. The application of the new transglutaminase-mediated ISH (TransISH) strategy to mRNA detection in mammalian tissues was demonstrated.
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Acknowledgments
We thank Dr. Masayuki Mitsumori of Hitachi Aloka Medical, Ltd. and Dr. Momoko Kitaoka for helpful discussions. This research was supported in part by the Japan Society for the Promotion of Science KAKENHI (Grant number 25289297).
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Miyawaki, K., Noji, S., Kamiya, N. (2015). Transglutaminase-Mediated In Situ Hybridization (TransISH) for mRNA Detection in Mammalian Tissues. In: Hauptmann, G. (eds) In Situ Hybridization Methods. Neuromethods, vol 99. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2303-8_29
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DOI: https://doi.org/10.1007/978-1-4939-2303-8_29
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