Detection of GPCR mRNA Expression in Primary Cells Via qPCR, Microarrays, and RNA-Sequencing.
Detection of GPCR mRNA Expression in Primary Cells Via qPCR, Microarrays, and RNA-Sequencing.
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DOI：
10.1007/978-1-0716-1221-7_2
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发表时间：
2021
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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.
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影响因子：
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作者：
Soneson C;Love MI;Robinson MD
通讯作者：
Robinson MD
影响因子：
9.8
作者：
Sriram, Krishna;Moyung, Kevin;Insel, Paul A.
通讯作者：
Insel, Paul A.
影响因子：
7.3
作者：
Alexander SP;Davenport AP;Kelly E;Marrion N;Peters JA;Benson HE;Faccenda E;Pawson AJ;Sharman JL;Southan C;Davies JA;CGTP Collaborators
通讯作者：
CGTP Collaborators
影响因子：
14.8
作者：
通讯作者：
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影响因子：
4.1
作者：
Sriram, Krishna;Wiley, Shu Z.;Insel, Paul A.
通讯作者：
Insel, Paul A.