Protocols for Localizing Receptor mRNAs and Proteins by RT-PCR, In Situ Hybridization, and Autoradiography

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Receptors, enzymes, transporters, and ion channels receive external signals and generate specific intracellular second messengers to mediate the biological response to the ligands that bind to the proteins on the cell membrane and/or to organelles inside the cells. In order to localize the potential target protein(s) that may be defective and/or those serving endogenous transmitter or drug activities in various tissues of the body, the use of reverse transcriptase-polymerase chain reaction and in situ hybridization coupled with quantitative receptor autoradiography are deployed. Such studies can be performed using isolated cells or postmortem tissues of animals and humans. Such techniques are powerful indicators of the presence or absence of the key receptors/enzymes and other protein targets that can help ensure target validation, localization, and engagement with exogenously delivered chemicals, drugs, antibodies, etc. These aspects are critical for drug discovery and can also indicate off-target side effects of drugs under study. These techniques will be described in this chapter.