Analysis of Gq protein alpha subunit mRNA expression in human monocytes: relevance of the purification step

Background: G alphaq is a member of the Gq family of G proteins, which by s timulating the phospholipase C beta (PLC beta)-IP3-Ca++ mediated intracellu lar signaling systems controls some of the most fundamental cardiovascular cellular processes. The study of G alphaq is complicated by the presence of a pseudogene in human DNA, with significant homology to the mRNA encoding the alpha subunit of Gq protein. The presence of this pseudogene will cause problems when the analysis of the G alphaq gene expression is based solely on RT-PCR, In this study, we report a simple method for avoiding unwanted amplification of the G alphaq pseudogene and the use of human monocytes as a readily available source for examining G alphaq. Methods: RT-PCR was carr ied out on RNA extracted from peripheral blood monocytes of 10 normal subje cts using specific primers for G alphaq. Results: When several subjects' G alphaq was examined, the authentic G alphaq mRNA amplification product leve ls, as a ratio to unpurified pseudogene containing amplification products, declined by up to approximately 70%. Conclusions: Given the importance of G q protein in cardiovascular signal transduction, it is fundamental to provi de a reliable assessment of Gag gene expression. In addition to accurately assessing G alphaq levels, the use of circulating human monocytes as a usef ul source of G alphaq for investigating mechanisms involved in the regulati on of vascular tone is shown. (C) 2001 Elsevier Science B.V. All rights res erved.