An innovative real-time PCR method to measure changes in RNA editing of the serotonin 2C receptor (5-HT2CR) in brain
An innovative real-time PCR method to measure changes in RNA editing of the serotonin 2C receptor (5-HT2CR) in brain
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DOI：
10.1016/j.jneumeth.2009.01.027
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发表时间：
2009-05-15
影响因子：
3
通讯作者：
Cunningham, Kathryn A.
中科院分区：
文献类型：
--
作者：
Lanfranco, Maria Fe;Seitz, Patricia K.;Cunningham, Kathryn A.
5-羟色胺2C受体（5-HT 2CR）在精神障碍（例如，抑郁症）并且是药物治疗的靶点。5-HT 2CR在脑和脊髓中广泛表达，是目前已知的唯一经历mRNA编辑的G蛋白偶联受体，mRNA编辑是一种转录后修饰，导致不同但密切相关的蛋白质同种型的翻译。5-HT 2CR RNA可以在五个位点进行编辑，以改变多达三个氨基酸，从而调节受体：G蛋白偶联和组成型活性。为了快速定量离体个体5-HT 2CR亚型水平响应于治疗的变化，我们采用了定量（实时）逆转录聚合酶链反应（qRT-PCR），该反应利用了经小沟结合剂（MGB）修饰的TaqMan（R）探针。开发了4种5-HT 2CR RNA亚型的探针，并使用标准模板系统地验证了其灵敏度和特异性。在从129 S6和C57 BL/6 J小鼠提取的全脑的cDNA中测量四种同种型的相对表达。从该qRT-PCR分析得出的排序与从DNA测序得出的排序相匹配。在仅表达未编辑或完全编辑的5-HT 2CR转录物的突变小鼠中，仅检测到预期的转录物。这些数据表明，这种qRT-PCR方法是一种精确和快速的方法，可以在体外检测密切相关的mRNA序列，而无需表征整个5-HT 2CR谱。这项技术的实施将扩大和加快特定的大脑5-HT 2CR mRNA异构体的研究，以响应药理学，行为和遗传操作，特别是在离体研究，需要快速收集大量的样本数据。(C)2009爱思唯尔有限公司版权所有。
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 (R) 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. (C) 2009 Elsevier B.V. All rights reserved.