Detection and Quantification of GPCR mRNA: An Assessment and Implications of Data from High-Content Methods
Detection and Quantification of GPCR mRNA: An Assessment and Implications of Data from High-Content Methods
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DOI：
10.1021/acsomega.9b02811
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发表时间：
2019-10-15
期刊：
影响因子：
4.1
通讯作者：
Insel, Paul A.
中科院分区：
文献类型：
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作者：
Sriram, Krishna;Wiley, Shu Z.;Insel, Paul A.
G 蛋白偶联受体 (GPCR) 是最大的膜受体家族，也是已批准药物的靶标。因此，GPCR 表达的分析对于药物发现非常重要，通常涉及基于信使 RN (mRNA) 的方法。我们比较了转录组互补 DNA (cDNA) (Affymetrix) 微阵列、RNA 测序 (RNA-seq) 和基于定量聚合酶链式反应 (qPCR) 的 TaqMan 阵列检测和定量 endGPCR（具有内源激动剂的非化学感应 GPCR）表达的能力。在人胰腺癌相关成纤维细胞中，RNA-seq 和 TaqMan 阵列产生了 GPCR 数量（接近 100）和表达水平密切相关的值，经独立 qPCR 验证。相比之下，微阵列未能识别出类似 30 个这样的 GPCR，并且生成的数据与这些方法的结果相关性较差。 RNA-seq 和 TaqMan 阵列还在人心脏成纤维细胞、胰腺星状细胞、癌细胞系和肺动脉平滑肌细胞中产生了类似的 GPCR 结果。几个 Gq/11 偶联 GPCR 的 mRNA 表达量预测了同源激动剂导致的胞浆钙增加和细胞迁移。 RNA-seq还揭示了endoGPCR的剪接变体。因此，基于 RNA-seq 和 qPCR 的阵列比转录组 cDNA 微阵列更适合评估 GPCR 表达，并且可以产生预测功能反应的结果，这些结果对 GPCR 生物学和药物发现具有影响。
G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and targets for approved drugs. The analysis of GPCR expression is, thus, important for drug discovery and typically involves messenger RN (mRNA)-based methods. We compared transcriptomic complementary DNA(cDNA) (Affymetrix) microarrays, RNA sequencing (RNA-seq), and quantitative polymerase chain reaction (qPCR)-based TaqMan arrays for their ability to detect and quantify expression of endoGPCRs (nonchemosensory GPCRs with endogenous agonists). In human pancreatic cancer-associated fibroblasts, RNA-seq and TaqMan arrays yielded closely correlated values for GPCR number (similar to 100) and expression levels, as validated by independent qPCR. By contrast, the microarrays failed to identify similar to 30 such GPCRs and generated data poorly correlated with results from those methods. RNA-seq and TaqMan arrays also yielded comparable results for GPCRs in human cardiac fibroblasts, pancreatic stellate cells, cancer cell lines, and pulmonary arterial smooth muscle cells. The magnitude of mRNA expression for several Gq/11-coupled GPCRs predicted cytosolic calcium increase and cell migration by cognate agonists. RNA-seq also revealed splice variants for endoGPCRs. Thus, RNA-seq and qPCR-based arrays are much better suited than transcriptomic cDNA microarrays for assessing GPCR expression and can yield results predictive of functional responses, findings that have implications for GPCR biology and drug discovery.