An innovative real-time PCR method to measure changes in RNA editing of the serotonin 2C receptor (5-HT2CR) in brain

摘要

The serotonin 2C receptor (5-HT2CR) plays a significant role in psychiatric disorders (e.g., depression) and is a target for pharmacotherapy. The 5-HT2CR is widely expressed in brain and spinal cord and is the only G-protein coupled receptor currently known to undergo mRNA editing, a post-transcriptional modification that results in translation of distinct, though closely related, protein isoforms. The 5-HT2CR RNA can be edited at five sites to alter up to three amino acids resulting in modulation of receptor:G-protein coupling and constitutive activity. To rapidly quantify changes ex vivo in individual 5-HT2CR isoform levels in response to treatment, we adapted quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) utilizing TaqMan^® probes modified with a minor groove binder (MGB). Probes were developed for four 5-HT2CR RNA isoforms and their sensitivity and specificity were validated systematically using standard templates. Relative expression of the four isoforms was measured in cDNAs from whole brain extracted from 129S6 and C57BL/6J mice. Rank order derived from this qRT-PCR analysis matched that derived from DNA sequencing. In mutant mice solely expressing either non-edited or fully edited 5-HT2CR transcripts, only expected transcripts were detected. These data suggest this qRT-PCR method is a precise and rapid means to detect closely related mRNA sequences ex vivo without the necessity of characterizing the entire 5-HT2CR profile. Implementation of this technique will expand and expedite studies of specific brain 5-HT2CR mRNA isoforms in response to pharmacological, behavioral and genetic manipulation, particularly in ex vivo studies which require rapid collection of data on large numbers of samples.