Sriram Krishna, Salmerón Cristina, Di Nardo Anna, Insel Paul A
Department of Pharmacology, University of California San Diego, La Jolla, CA, USA.
Department of Dermatology, University of California San Diego, La Jolla, CA, USA.
Methods Mol Biol. 2021;2268:21-42. doi: 10.1007/978-1-0716-1221-7_2.
A workflow is described for assaying the expression of G protein-coupled receptors (GPCRs) in cultured cells, using a combination of methods that assess GPCR mRNAs. Beginning from the isolation of cDNA and preparation of mRNA, we provide protocols for designing and testing qPCR primers, assaying mRNA expression using qPCR and high-throughput analysis of GPCR mRNA expression via TaqMan qPCR-based, GPCR-selective arrays. We also provide a workflow for analysis of expression from RNA-sequencing (RNA-seq) assays, which can be queried to yield expression of GPCRs and related genes in samples of interest, as well as to test changes in expression between groups, such as in cells treated with drugs or from healthy and diseased subjects. We place priority on optimized protocols that distinguish signal from noise, as GPCR mRNAs are typically present in low abundance, necessitating techniques that maximize sensitivity while minimizing noise. These methods may also be applicable for assessing the expression of members of families of other low abundance genes via high-throughput analyses of mRNAs, followed by independent confirmation and validation of results via qPCR.
本文描述了一种用于检测培养细胞中G蛋白偶联受体（GPCR）表达的工作流程，该流程结合了多种评估GPCR mRNA的方法。从cDNA的分离和mRNA的制备开始，我们提供了设计和测试qPCR引物的方案，使用qPCR检测mRNA表达，以及通过基于TaqMan qPCR的GPCR选择性阵列对GPCR mRNA表达进行高通量分析。我们还提供了一种用于分析RNA测序（RNA-seq）检测结果的工作流程，该流程可用于查询感兴趣样本中GPCR和相关基因的表达情况，以及测试不同组之间的表达变化，例如用药物处理的细胞或健康和患病受试者的细胞。我们优先考虑优化的方案，以区分信号与噪声，因为GPCR mRNA通常以低丰度存在，这就需要采用能在最大限度提高灵敏度的同时最小化噪声的技术。这些方法也可用于通过mRNA的高通量分析来评估其他低丰度基因家族成员的表达，随后通过qPCR对结果进行独立确认和验证。
