Analysis of Gq protein alpha subunit mRNA expression in human monocytes: relevance of the purification step.
BACKGROUND
Galphaq is a member of the Gq family of G proteins, which by stimulating the phospholipase Cbeta (PLCbeta)-IP(3)-Ca(++) mediated intracellular signaling systems controls some of the most fundamental cardiovascular cellular processes. The study of Galphaq is complicated by the presence of a pseudogene in human DNA, with signficant homology to the mRNA encoding the alpha subunit of Gq protein. The presence of this pseudogene will cause problems when the analysis of the Galphaq gene expression is based solely on RT-PCR. In this study, we report a simple method for avoiding unwanted amplification of the Galphaq pseudogene and the use of human monocytes as a readily available source for examining Galphaq.


METHODS
RT-PCR was carried out on RNA extracted from peripheral blood monocytes of 10 normal subjects using specific primers for Galphaq.


RESULTS
When several subjects' Galphaq was examined, the authentic Galphaq mRNA amplification product levels, as a ratio to unpurified pseudogene containing amplification products, declined by up to approximately 70%.


CONCLUSIONS
Given the importance of Gq protein in cardiovascular signal transduction, it is fundamental to provide a reliable assessment of G alpha q gene expression. In addition to accurately assessing Galphaq levels, the use of circulating human monocytes as a useful source of Galphaq for investigating mechanisms involved in the regulation of vascular tone is shown.
Calò
L
L
Department of Clinical and Experimental Medicine, Clinica Medica 4, University of Padova-Azienda Ospedaliera, Via Giustiniani, 2, 35128, Padua, Italy. renzcalo@unipd.it
Milani
M
M
Pagnin
E
E
Davis
P
P
Piccin
A
A
Costa
R
R
Plebani
M
M
Semplicini
A
A
D001483:Base Sequence; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D015870:Gene Expression; D020962:Heterotrimeric GTP-Binding Proteins / Q000097:blood* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D009000:Monocytes / Q000737:chemistry* / Q000166:cytology; D011544:Pseudogenes / Q000235:genetics*; D012333:RNA, Messenger / Q000097:blood*; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
D012333:RNA, Messenger; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins