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1 Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland; and the 2 Departments of Medicine and Pathology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
In recent years, both molecular biological and immunohistochemical techniques, utilizing receptor subtype-specific probes and antibodies to cloned central nervous system dopamine receptors, have revealed their presence in a number of peripheral organs and tissues. Molecular techniques have been hindered by the low abundance of receptor mRNA in these sites, and reverse transcription-polymerase chain reaction (RT-PCR) has been utilized to address this problem. However, RT-PCR is most often employed on either isolated mRNA or microdissected tissue samples, thereby limiting interpretation of whole tissue distribution. The present paper describes the use of a novel self-sustained sequence replication system (3SR) to amplify a target mRNA sequence in situ within the tissue or cell of interest that is then detected with the use of an internal labeled probe, using standard nonisotopic in situ hybridization. Specifically, D1A receptor mRNA was amplified and detected in kidney sections of Wistar-Kyoto rats (WKY). The amplified D1A receptor mRNA was localized to renal arterioles, juxtaglomerular apparatus, and both proximal and distal tubules. mRNA was colocalized to regions shown also to contain D1A receptor protein. D1A receptor mRNA was predominantly localized in the cortex. Specificity of D1A receptor mRNA detection was confirmed by appropriate localization in rat brain sections known to express D1A receptor mRNA. In addition, we confirmed the presence of renal D1A receptor mRNA by RT-PCR. We conclude that D1A receptor mRNA is expressed in a site-specific manner in the WKY kidney. The use of 3SR in situ permits elucidation of site specific mRNA localization in a manner not reported previously.
in situ amplification; dopamine D1A receptor; messenger ribonucleic acid; kidney; in situ hybridization
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