Optimization and standardization of circulating microRNA isolation from patients with Familial Hypercholesterolemia

Abstract

Epigenetic is the study of reversible and inherited modifications during cell division that can change the final biological activity of proteins without DNA sequence changes. microRNA (miRNA) is an epigenetic mechanism which control the gene expression by translation inhibition or messenger RNA degradation. Differential expression of miRNA has already been associated with cardiovascular and metabolic diseases, including dyslipidemias. However, the elucidation of the role of miRNA in the development of these diseases may contribute to the discovery of new biomarkers, as well as to the discovery of targets for the development of new drugs. miRNA have been identified as key molecules for the regulation of protein synthesis, such as those encoded by the genes SREBP1, SREBP2, ABCA1, LDLR, HMGCR and APOA1 involved in cholesterol homeostasis. However, many studies are still necessary to understand the influence of miRNA on the pathophysiology of Familial Hypercholesterolemia (FH). This work plan aims to standardize miRNA isolation from plasma of patients with FH. These miRNA will be used in other steps of the study. For this, plasma collected from patients with tube contain EDTA as an anticoagulant will be used for miRNA isolation using the miRNeasy Serum / Plasma kit (Qiagen). First, the protocol will be performed according to the manufacturer's recommendations. Then, different plasma inputs will be evaluated and adaptations will be made to increase miRNA yield, which will be evaluated through the expression analysis of an exogenous control (mir-39) by quantitative Real-Time PCR. miRNA will be stored at -80ºC until the next steps of study.