Identification of Reference Genes for Quantitative RT-PCR in Ascending Aortic Aneurysms

Abstract

Hypertension and congenital aortic valve malformations are frequent causes of ascending aortic aneurysms. The molecular mechanisms of aneurysm formation under these circumstances are not well understood. Reference genes for gene activity studies in aortic tissue that are not influenced by aortic valve morphology and its hemodynamic consequences, aortic dilatation, hypertension, or antihypertensive medication are not available so far. This study determines genes in ascending aortic tissue that are independent of these parameters. Tissue specimens from dilated and undilated ascending aortas were obtained from 60 patients (age ≤70 years) with different morphologies of the aortic valve (tricuspid undilated n = 24, dilated n = 11; bicuspid undilated n = 6, dilated n = 15; unicuspid dilated n = 4). Of the studied individuals, 36 had hypertension, and 31 received ACE inhibitors or AT1 receptor antagonists. The specimens were obtained intraoperatively from the wall of the ascending aorta. We analyzed the expression levels of 32 candidate reference genes by quantitative RT-PCR (RT-qPCR). Differential expression levels were assessed by parametric statistics. The expression analysis of these 32 genes by RT-qPCR showed that EIF2B1, ELF1, and PPIA remained constant in their expression levels in the different specimen groups, thus being insensitive to aortic valve morphology, aortic dilatation, hypertension, and medication with ACE inhibitors or AT1 receptor antagonists. Unlike many other commonly used reference genes, the genes EIF2B1, ELF1, and PPIA are neither confounded by aortic comorbidities nor by antihypertensive medication and therefore are most suitable for gene expression analysis of ascending aortic tissue.

Hypothesis-free secretome analysis of thoracic aortic aneurysm reinforces the central role of TGF-β cascade in patients with bicuspid aortic valve

“…PCR conditions and primer sequences used were previously described [11]. Following recent guidelines PPIA [12], beta-actin, and GADPH were used as candidate reference genes.…”

Selective and marked decrease of complement receptor C5aR2 in human thoracic aortic aneurysms: a dysregulation with potential inflammatory effects

“…Quantification of gene expression was performed in duplicate by quantitative real-time PCR using Taqman probes (See online supplementary material table S3). Gene expression was normalised against EIF2β1, a gene highly suitable for TAA materials,20 and relative gene expression calculated using the 2 -ΔΔCt -method.…”

Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve

“…β-Actin , G3pdh , β2m ) are commonly used with this purpose. However, numerous studies have reported that the expression levels of classical reference genes are not stable in many tissues under certain physiological conditions, making them inadequate for gene expression normalization [ 19 , 22 – 25 ]. Accordingly, it is recognized that, in order to obtain reliable data using RT-qPCR analyses, the stability of the housekeeping gene chosen for the experimental setup must be previously demonstrated.…”

“…A key aspect of the method is the normalization of mRNA concentrations with housekeeping or reference genes [ 18 , 19 ], the stability of which must be confirmed in the tissue employed in order to obtain accurate data [ 18 , 20 , 21 ]. However, several common reference genes are frequently used in RT-qPCR assays without further validation, even though they have been found unstable in different tissues and physiological conditions [ 19 , 22 – 24 ], including the dilated ascending aorta [ 25 ].…”