Balkan Journal of Medical Genetics

A PRELIMINARY microRNA ANALYSIS OF NON SYNDROMIC THORACIC AORTIC ANEURYSMS
Patuzzo C1,*, Pasquali A1, Malerba G1, Trabetti E1, Pignatti PF1, Tessari M2, Faggian G2
*Corresponding Author: Dr. Cristina Patuzzo, Department of Life and Reproduction Sciences, University of Verona, Strada Le Grazie 8, 37134,Verona, Italy; Tel.: +39-45-802-7207; Fax: +39-45-802-7180; E-mail: cristina.patuzzo@univr.it
page: 51

MATERIALS AND METHODS

Patients and Biological Samples. Samples of aneurysmal ascending aortic wall were obtained from 10 patients (five males and five females) affected by non familial non syndromic TAA, during surgical repair of their ascending aneurysms. The syndromic nature of aortic aneurysm has been systematically excluded by careful evaluation of clinical and family history. Patients with aortic dissections, ruptured aneurysms, Marfan syndrome, or other known connective tissue disorders, were excluded from the study. Autoimmune and/or infectious inflammatory diseases, or chest trauma, were also excluded. Control samples of ascending aorta were obtained from 10 heart transplant recipients without aortic aneurysms (five males and five females). All the individuals had a tricuspid aortic valve. Mean age was 66 ± 9 years. The study conforms with the principles outlined in the Declaration of Helsinki. Aortic samples were promptly dipped into RNAlater solution (Ambion, Austin, TX, USA) in order to preserve their cellular RNA and maintained at room temperature for 2 hours to facilitate liquid permeation. The samples were then stored at –80°C. Preparation of Microarrays. Total RNA was extracted from the 20 aorta specimens with TRIzol reagent according to the manufacturer’s protocol. Total RNA integrity was assessed by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA integrity numbers (RIN) were sufficient for micro RNA (miRNA) microarray experiments (i.e., RIN >6) [11]. Four samples were then prepared by pooling corresponding RNAs: TAA males, TAA females, control males, and control females. Sample labeling, PIQOR™ mirExplore microarray hybridization and fluorescence signal detection were performed by Miltenyi Biotec GmbH (MACS Service, Köln, Germany). The TAA and control pools were labeled with Hy5 and Hy3, respectively, and competitively hybridized on the same microarray, separately for the two sexes. Fluorescence signals of the hybridized PIQOR™ Microarrays (Miltenyi Biotec GmbH) were detected using a laser scanner from Agilent (Agilent Technologies). Image and Data Analysis. Mean signal and mean local background intensities were obtained for each spot of the microarray images using the ImaGene® software (Biodiscovery, Hawthorne, CA, USA). Low-quality spots were flagged and excluded from data analysis. Unflagged spots were analyzed with the PIQOR™ Analyzer software (Miltenyi Biotec GmbH) that allows automated data processing of the raw data text files derived from the ImaGene software. This includes background subtraction to obtain the net signal intensity, data normalization, and calculation of the Hy5/Hy3 ratios. As an additional quality filtering step, only spots/genes that had a signal higher than the 50.0% percentile of the background signal intensities were taken into account for the calculation of the Hy5/ Hy3 ratio. Up- and Down-Regulated MicroRNA and Gene Pathways. Normalized mean Hy5/Hy3 ratios were determined for four replicas per gene. There was a specific detection even for closely related miRNA family members. MicroRNAs that were >1.5-fold-up or down-regulated represented putative candidate miRNAs. To identify molecular pathways potentially altered by the expression of single or multiple miRNAs, Diana mir- Path Software (Athens, Greece) was used [12]. This web-based application performs an enrichment analysis of multiple miRNA target genes comparing each set of miRNA targets to all known KEGG (Kyoto Encyclopedia of Genes and Genomes, Kyoto, Japan) pathways [13].

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