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

Lanfranco MF, Seitz PK, Morabito MV, Emeson RB, Sanders-Bush E, Cunningham KA
J Neurosci Methods. 2009 179 (2): 247-57

PMID: 19428534 · PMCID: PMC3794365 · DOI:10.1016/j.jneumeth.2009.01.027

The serotonin 2C receptor (5-HT(2C)R) plays a significant role in psychiatric disorders (e.g., depression) and is a target for pharmacotherapy. The 5-HT(2C)R 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-HT(2C)R 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-HT(2C)R 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-HT(2C)R 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-HT(2C)R 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-HT(2C)R profile. Implementation of this technique will expand and expedite studies of specific brain 5-HT(2C)R 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.

MeSH Terms (18)

Alternative Splicing Animals Base Sequence Brain Gene Expression Profiling Male Mice Mice, Inbred C57BL Mutation Protein Isoforms Receptor, Serotonin, 5-HT2C Reverse Transcriptase Polymerase Chain Reaction RNA, Messenger RNA Editing RNA Processing, Post-Transcriptional Sensitivity and Specificity Serotonin Time Factors

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