Optimisation of methods for quantifying plasma mRNA levels from genes responsible for coronary artery plaque development and destabilization.
OBJECTIVE
To investigate the hypothesis that in patients with coronary atherosclerosis it is possible to measure plasma mRNA levels from genes responsible for plaque development and destabilization.


METHODS
Methods for RNA isolation, mRNA transcription and quantitative PCR were evaluated and optimised, in order to achieve reliable mRNA quantification. RESULTS mRNA level was possible to quantify from plasma of patients with coronary atherosclerosis, as well as from healthy volunteers, from genes encoding cathepsin S, cathepsin B, CD40 molecule, monocyte chemotactic protein 1, death-associated protein kinase 1, matrix metallopeptidase 9, vascular cell adhesion molecule 1 and phosphoglycerate kinase 1 (reference gene). Analytical between-run imprecision of average threshold cycle, expressed as coefficient of variation was below 2%. EDTA blood samples should be centrifuged within one hour of venesection. It was not possible to quantify plasma mRNA level from genes encoding macrophage scavenger receptor 1, perilipin, tissue factor, phospholipase A2 group IIA, collagen type I alpha 2 and interleukin 1 alpha.


CONCLUSIONS
Further plasma mRNA analysis is reasonable to access its potential usefulness in non-invasive in vivo monitoring of gene expression profile in vascular beds.
Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia. darko.cerne@ffa.uni-Ij.si
Darko
D
Cerne
Irma
I
Stern
Igor
I
Kranjec
Janja
J
Marc
D003324:Coronary Artery Disease / Q000097:blood / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D010949:Plasma / Q000737:chemistry*; D012333:RNA, Messenger / Q000097:blood* / Q000302:isolation & purification
D012333:RNA, Messenger