Amisten Stefan
Diabetes Research Group, King's College London, London, UK.
Methods Cell Biol. 2016;132:73-105. doi: 10.1016/bs.mcb.2015.10.004. Epub 2015 Dec 24.
G protein-coupled receptors (GPCRs) are important regulators of human physiology and therefore the targets of a large number of modern therapeutics. Although GPCRs are important regulators of adipose tissue endocrine and energy storage functions, the expression and function of a majority of GPCRs in adipose tissue is poorly characterized. A first step in the functional characterization of adipose tissue GPCRs is to accurately quantify the expression of GPCRs in adipose tissue. In this methods chapter, a detailed, step-by-step protocol is presented for the isolation of adipose tissue total RNA, its conversion into cDNA and the real-time PCR quantification of human GPCR mRNA expression relative to the mRNA expression of the stable adipose tissue housekeeping gene peptidylprolyl isomerase A (PPIA). A comprehensive list of 377 manually validated, commercially available GPCR qPCR primers allows facilitated swift quantification of either the entire human GPCRome or individual GPCRs, thus providing a sensitive, flexible, and cost-effective means of determining the mRNA expression of GPCRs in adipose tissue.
G蛋白偶联受体（GPCRs）是人类生理学的重要调节因子，因此也是大量现代治疗药物的靶点。尽管GPCRs是脂肪组织内分泌和能量储存功能的重要调节因子，但大多数GPCRs在脂肪组织中的表达和功能仍未得到充分表征。脂肪组织GPCRs功能表征的第一步是准确量化脂肪组织中GPCRs的表达。在本章方法中，我们将详细介绍从脂肪组织中分离总RNA、将其转化为cDNA以及相对于稳定的脂肪组织管家基因肽基脯氨酰异构酶A（PPIA）的mRNA表达进行人GPCR mRNA表达实时定量PCR的一步步操作流程。一份包含377种经过人工验证的市售GPCR qPCR引物的综合列表，有助于快速、便捷地对整个人类GPCR组或单个GPCR进行定量，从而提供一种灵敏、灵活且经济高效的方法来测定脂肪组织中GPCRs的mRNA表达。
