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Comparative analysis of methods to profile mRNA expression of G‐protein coupled receptors
Author(s) -
Sriram Krishna,
Zhou Shu,
Lowy Andrew M.,
Insel Paul A.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1028.4
Subject(s) - g protein coupled receptor , taqman , biology , computational biology , dna microarray , microarray , messenger rna , receptor , gene expression profiling , gene , gene expression , microbiology and biotechnology , bioinformatics , genetics , real time polymerase chain reaction
G‐Protein coupled receptors (GPCRs) comprise the largest family of cell surface receptors and proteins used as targets of approved drugs. Due to their low abundance, paucity of specific and sensitive antibodies and the challenge of identifying receptors in individual cell types, efforts to profile GPCR expression typically involve mRNA‐based methods and the relative quantification of gene expression. In the current study we compared in parallel the use of Affymetrix microarrays, RNAseq and qPCR‐based arrays for the measurement of mRNA expression of non‐chemosensory endogenously expressed (endo)GPCRs. We analyzed samples of patient‐derived pancreatic cancer‐associated fibroblasts (CAFs). RNAseq was done with a read depth of ~25 million reads/sample with single‐end 75bp reads. Affymetrix samples were run on the GeneChip Human Genome U133 Plus 2.0 Array chip and for qPCR, we used Taqman GPCR arrays from Life Technologies. Our results demonstrate a high degree of correlation between results obtained from the GPCR arrays and RNAseq. In general, the number of GPCRs detected (~100 per sample), their identity and magnitude of expression were closely correlated with those 2 methods. The validity of the results was verified by independent qPCR. By comparison, the Affymetrix arrays failed to identify a large number of GPCRs (~30) detected by both RNAseq and Taqman arrays. In addition, different probe sets for the same GPCR on Affymetrix arrays sometimes resulted in drastically divergent mRNA intensities, even though both values were reported as being “statistically significant” (P<0.05). We conclude that RNAseq and qPCR‐based arrays both provide appropriate methods to assess GPCR expression (although each method has advantages and disadvantages) and are preferable to Affymetrix arrays for the detection of GPCR mRNAs, especially low abundance GPCRs. Importantly, our mRNA analyses of GPCR expression by both RNA seq and GPCR arrays indicate that CAFs and other individual cell types express >100 different GPCRS, including a large number of orphan (without known physiologic agonists) receptors, certain of which we find can regulate cell function and may be novel drug targets. Support or Funding Information Supported by NIH R21CA189477 and T32CA121938