Optimisation of methods for quantifying plasma mRNA levels from genes responsible for coronary artery plaque development and destabilization

Abstract

Aim To investigate the hypothesis that in patients with coronaryatherosclerosis it is possible to measure plasma mRNA levels fromgenes responsible for plaque development and destabilization.Methods Methods for RNA isolation, mRNA transcription and quantitative PCR were evaluated and optimised, in order to achievereliable mRNA quantiication. 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 coeficient of variation was below 2%. EDTA blood samples should be centrifuged within one hour of venesection. It wasnot possible to quantify plasma mRNA level from genes encodingmacrophage scavenger receptor 1, perilipin, tissue factor, phospholipase A2 group IIA, collagen type I alpha 2 and interleukin 1alpha. Conclusion Further plasma mRNA analysis is reasonable to access its potential usefulness in non-invasive in vivo monitoring ofgene expression proile in vascular beds.

Keywords

• plasma mRNA
• coronary atherosclerosis
• method optimisation