Selection of reference genes for quantitative real time PCR (qPCR) assays in tissue from human ascending aorta.
Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples.
UGC del Corazón, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Clínico Universitario Virgen de la Victoria, Málaga, Spain.
Carmen
C
Rueda-Martínez
UGC del Corazón, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Clínico Universitario Virgen de la Victoria, Málaga, Spain.
Oscar
O
Lamas
UGC del Corazón, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Clínico Universitario Virgen de la Victoria, Málaga, Spain.
María José
MJ
Mataró
UGC del Corazón, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Clínico Universitario Virgen de la Victoria, Málaga, Spain.
Juan
J
Robledo-Carmona
UGC del Corazón, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Clínico Universitario Virgen de la Victoria, Málaga, Spain.
Gemma
G
Sánchez-Espín
UGC del Corazón, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Clínico Universitario Virgen de la Victoria, Málaga, Spain; Departamento de Medicina, Facultad de Medicina, Universidad de Málaga, Málaga, Spain.
Manuel
M
Jiménez-Navarro
UGC del Corazón, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Clínico Universitario Virgen de la Victoria, Málaga, Spain.
Miguel
M
Such-Martínez
Departamento de Biología Animal, Instituto de Investigación Biomédica de Málaga (IBIMA), Facultad de Ciencias, Universidad de Málaga, Málaga, Spain.
Borja
B
Fernández
D000368:Aged; D000465:Algorithms; D001011:Aorta / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics; D004108:Dilatation, Pathologic / Q000378:metabolism; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D016601:RNA-Binding Proteins; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
C459802:CASC3 protein, human; C436291:CDKN1B protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27