title: Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-α mRNA expression.
abstract: Real-time PCR is the benchmark method for measuring mRNA expression levels, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. Though the minipig model is largely used to study cardiovascular disease, no specific reference genes have been identified in porcine myocardium. The aim of the study was to identify and validate reference gene to be used in RT-PCR studies of failing (HF) and non-failing pig hearts. Eight candidate reference genes (GAPDH, ACTB, B2M, TBP, HPRT-1, PPIA, TOP2B, YWHAZ) were selected to compare cardiac tissue of normal (n=4) and HF (n=5) minipigs. The most stable genes resulted: HPRT-1, TBP, PPIA (right and left atrium); PPIA, GAPDH, ACTB (right ventricle); HPRT-1, TBP, GAPDH (left ventricle). The normalization strategy was tested analyzing mRNA expression of TNF-α, which is known to be up-regulated in HF and whose variations resulted more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance to provide a set of reference genes to normalize mRNA expression in HF and control minipigs. The use of unvalidated reference genes can generate biased results because also their expression could be altered by the experimental conditions.
authors.affiliation: University of Pisa, Department of Biology, Genetics Division, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Martino
authors.forename: Manuela
authors.initials: M
authors.lastname: Cabiati
authors.forename: Manuela
authors.initials: M
authors.lastname: Campan
authors.forename: Tommaso
authors.initials: T
authors.lastname: Prescimone
authors.forename: Daiana
authors.initials: D
authors.lastname: Minocci
authors.forename: Chiara
authors.initials: C
authors.lastname: Caselli
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Rossi
authors.forename: Daniela
authors.initials: D
authors.lastname: Giannessi
authors.forename: Silvia
authors.initials: S
authors.lastname: Del Ry
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D004195:Disease Models, Animal; D020869:Gene Expression Profiling; D005819:Genetic Markers / Q000235:genetics*; D006325:Heart Atria / Q000737:chemistry / Q000378:metabolism; D006333:Heart Failure / Q000235:genetics* / Q000378:metabolism; D006352:Heart Ventricles / Q000737:chemistry / Q000378:metabolism; D008297:Male; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D013552:Swine; D013556:Swine, Miniature; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha