Optimization of human 14 000 oligonucleotide-microarrays and study design for the evaluation of gene expression profiling in cardiovascular diseases

Abstract number: P0675

Giusti B., Gori A. M., Rossi L., Bandinelli B., Lucarini L., Evangelisti L., Lapini I., Lenti M., Abbate R., Pepe G.

University of Florence, Italy

Cardiovascular diseases (CVDs) is a leading cause of death. CVDs result from mechanisms involving well known environmental factors and genetic factors. Our group is involved in the comprehension of the physiopathological mechanisms of aortic aneurysms. Clinical and experimental evidences demonstrate an association among atherosclerotic lesions and inflammation. A clinical expression of the atherosclerosis are dilatations or aneurysms resulting from chronic inflammations and extracellular matrix degradation. Although these processes are considered critical in the pathophysiology of aneurysmal degeneration, the exact causes of aneurysm growth and rupture are still unknown. In recent years, the development of microarrays technology has made it possible to examine the simultaneous expression of multiple (thousand) gene products in the same experiment. These methods greatly facilitate the identification of altered patterns of gene expression including detection of unanticipated changes. In order to obtain a global portrait of gene expression in syndromic (Marfan syndrome) and non syndromic abdominal and thoracic aortic aneurysms, we choose the oligonucleotide-microarrays approach. We used the 14 000 genes human bank (70-mers oligonucleotides) to construct our arrays. Full-thickness aortic well specimens, fibroblast culture from skin biopsy and venous peripheral blood samples were obtained from 18 patients undergoing elective surgical repair (6 Marfan, 7 Abdominal Aortic Aneurysm and 5 Thoracic Aortic Aneurysm). We also enrolled sex and age matched controls. Aortic specimens were collected and stored in ‘RNA later’ solution before undergoing total RNA extraction. Total RNA from tissues and fibroblasts was isolated with Rneasy Kit (QIAGEN). Venous peripheral blood samples were collected in PAX gene Blood RNA tubes and total RNA was isolated with the ‘PAX gene RNA’ Kit (PreAnalyticX). Pooling of total RNA from patients and control subjects was performed in order to reduce individual genetic variability. During the optimization of the protocols we observed that the better data were obtained performing a 6-h hybridization of oligonucleotide-microarrays with 25 mg of total RNA. We are now evaluating data obtained from fibroblasts and peripheral blood cells of patients and controls. We expect that the mRNA expression profile analysis by microarrays will give as new insite on the wide molecular perturbation underlying these cardiovascular disorders.