Detection of mRNA by non-radioactive direct primed in situ reverse transcription (original) (raw)

Abstract.

There are various techniques to detect mRNA in tissue specimens. Among these in situ hybridization is widely applied, and for the detection of small quantities of RNA in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR) has been applied. Furthermore in situ transcription, where signal is produced by direct incorporation of labeled nucleotides during production of a cDNA by reverse transcription, has been shown by a few investigators. We present a non-radioactive in situ reverse transcriptase (in situ RT) protocol which is at least as sensitive as in situ hybridization but avoids probe production and long procedures of preincubation, incubation, and washing. Digoxigenin-labeled UTP is incorporated into a cDNA produced by in situ reverse transcription of mRNA. This method is combined with the fast and sensitive immunogold-silver detection system allowing demonstration of the mRNA within 7 h compared to days in the case of in situ hybridization. Contrary to in situ RT-PCR this new method of in situ RT has no background problems due to non-specific amplification or diffusion of the reaction product.

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Authors and Affiliations

1. Institute of Pathology, Johannes Gutenberg University, Langenbeckstrasse 1, 55101 Mainz, Germany, Germany
   Jörg Kriegsmann, Mike Otto & Torsten Hansen
2. Department of Internal Medicine I, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany, Germany
   Ulf Müller-Ladner
3. Centre for Experimental Rheumatology, Department of Rheumatology, University Hospital, Gloriastrasse 25, 8091 Zürich, Switzerland, Switzerland
   Haiko Sprott, Renate E. Gay & Steffen Gay
4. Institute of Pathology, Friedrich Schiller University, Ziegelmühlenweg 1, 07740 Jena, Germany, Germany
   Rolf Bräuer & Peter K. Petrow

Authors

1. Jörg Kriegsmann
2. Ulf Müller-Ladner
3. Haiko Sprott
4. Rolf Bräuer
5. Peter K. Petrow
6. Mike Otto
7. Torsten Hansen
8. Renate E. Gay
9. Steffen Gay

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Kriegsmann, J., Müller-Ladner, U., Sprott, H. et al. Detection of mRNA by non-radioactive direct primed in situ reverse transcription.Histochem Cell Biol 116, 199–204 (2001). https://doi.org/10.1007/s004180100295

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• Accepted: 22 May 2001
• Published: 11 August 2001
• Issue date: September 2001
• DOI: https://doi.org/10.1007/s004180100295