An innovative real-time PCR method to measure changes in RNA editing of the serotonin 2C receptor (5-HT(2C)R) in brain.
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.
Lanfranco
Maria Fe
MF
Center for Addiction Research and Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Route 0616, Galveston, TX 77555-0616, USA.
Seitz
Patricia K
PK
Morabito
Michael V
MV
Emeson
Ronald B
RB
Sanders-Bush
Elaine
E
Cunningham
Kathryn A
KA
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D001921:Brain / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics; D017393:RNA Editing / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D044405:Receptor, Serotonin, 5-HT2C / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012701:Serotonin / Q000378:metabolism; D013997:Time Factors
D020033:Protein Isoforms; D012333:RNA, Messenger; D044405:Receptor, Serotonin, 5-HT2C; D012701:Serotonin