title: Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation.
abstract: MicroRNAs (miRNAs) are emerging as key regulators of complex biological processes in several cardiovascular diseases, including atrial fibrillation (AF). Reverse transcription-quantitative polymerase chain reaction is a powerful technique to quantitatively assess miRNA expression profile, but reliable results depend on proper data normalization by suitable reference genes. Despite the increasing number of studies assessing miRNAs in cardiac disease, no consensus on the best reference genes has been reached. This work aims to assess reference genes stability in human cardiac tissue with a focus on AF investigation. We evaluated the stability of five reference genes (U6, SNORD48, SNORD44, miR-16, and 5S) in atrial tissue samples from eighteen cardiac-surgery patients in sinus rhythm and AF. Stability was quantified by combining BestKeeper, delta-Cq, GeNorm, and NormFinder statistical tools. All methods assessed SNORD48 as the best and U6 as the worst reference gene. Applications of different normalization strategies significantly impacted miRNA expression profiles in the study population. Our results point out the necessity of a consensus on data normalization in AF studies to avoid the emergence of divergent biological conclusions.
authors.affiliation: Department of Physics, University of Trento, Trento, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Masè
authors.affiliation: Centre for Integrative Biology, University of Trento, Trento, Italy.
authors.forename: Margherita
authors.initials: M
authors.lastname: Grasso
authors.affiliation: Department of Physics, University of Trento, Trento, Italy.|Centre for Integrative Biology, University of Trento, Trento, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Avogaro
authors.affiliation: Department of Physics, University of Trento, Trento, Italy.
authors.forename: Elvira
authors.initials: E
authors.lastname: D'Amato
authors.affiliation: Healthcare Research and Innovation Program (IRCS-PAT), Bruno Kessler Foundation, Trento, Italy.|Department of Industrial Engineering, University of Trento, Trento, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Tessarolo
authors.affiliation: Division of Cardiac Surgery, Santa Chiara Hospital, Trento, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Graffigna
authors.affiliation: Centre for Integrative Biology, University of Trento, Trento, Italy.
authors.forename: Michela Alessandra
authors.initials: MA
authors.lastname: Denti
authors.affiliation: Department of Physics, University of Trento, Trento, Italy.
authors.forename: Flavia
authors.initials: F
authors.lastname: Ravelli
mesh_terms: D000368:Aged; D001281:Atrial Fibrillation / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D009206:Myocardium / Q000378:metabolism*; D012015:Reference Standards
chemical_list: D035683:MicroRNAs