title: Selection of reference genes in different myocardial regions of an in vivo ischemia/reperfusion rat model for normalization of antioxidant gene expression.
abstract: BACKGROUND
  Changes in cardiac gene expression due to myocardial injury are usually assessed in whole heart tissue. However, as the heart is a heterogeneous system, spatial and temporal heterogeneity is expected in gene expression.
  
  
  RESULTS
  In an ischemia/reperfusion (I/R) rat model we evaluated gene expression of mitochondrial and cytoplasmatic superoxide dismutase (MnSod, Cu-ZnSod) and thioredoxin reductase (trxr1) upon short (4 h) and long (72 h) reperfusion times in the right ventricle (RV), and in the ischemic/reperfused (IRR) and the remote region (RR) of the left ventricle. Gene expression was assessed by Real-time reverse-transcription quantitative PCR (RT-qPCR). In order to select most stable reference genes suitable for normalization purposes, in each myocardial region we tested nine putative reference genes by geNorm analysis. The genes investigated were: Actin beta (actb), Glyceraldehyde-3-P-dehydrogenase (gapdh), Ribosomal protein L13A (rpl13a), Tyrosine 3-monooxygenase (ywhaz), Beta-glucuronidase (gusb), Hypoxanthine guanine Phosphoribosyltransferase 1 (hprt), TATA binding box protein (tbp), Hydroxymethylbilane synthase (hmbs), Polyadenylate-binding protein 1 (papbn1). According to our findings, most stable reference genes in the RV and RR were hmbs/hprt and hmbs/tbp/hprt respectively. In the IRR, six reference genes were recommended for normalization purposes; however, in view of experimental feasibility limitations, target gene expression could be normalized against the three most stable reference genes (ywhaz/pabp/hmbs) without loss of sensitivity. In all cases MnSod and Cu-ZnSod expression decreased upon long reperfusion, the former in all myocardial regions and the latter in IRR alone. trxr1 expression did not vary.
  
  
  CONCLUSIONS
  This study provides a validation of reference genes in the RV and in the anterior and posterior wall of the LV of cardiac ischemia/reperfusion model and shows that gene expression should be assessed separately in each region.
authors.affiliation: Istituto di Fisiologia Clinica, Consiglio Nazionale delle Ricerche, Pisa, Italy. nvesenti@ifc.cnr.it
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Vesentini
authors.forename: Cristina
authors.initials: C
authors.lastname: Barsanti
authors.forename: Alessandro
authors.initials: A
authors.lastname: Martino
authors.forename: Claudia
authors.initials: C
authors.lastname: Kusmic
authors.forename: Andrea
authors.initials: A
authors.lastname: Ripoli
authors.forename: AnnaMaria
authors.initials: A
authors.lastname: Rossi
authors.forename: Antonio
authors.initials: A
authors.lastname: L'Abbate
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D004195:Disease Models, Animal; D020869:Gene Expression Profiling / Q000592:standards*; D005966:Glucuronidase / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000235:genetics*; D009206:Myocardium / Q000378:metabolism*; D039122:Poly(A)-Binding Protein I / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics; D013482:Superoxide Dismutase / Q000235:genetics*; D000072105:Superoxide Dismutase-1; D035181:TATA-Box Binding Protein / Q000235:genetics; D054481:Thioredoxin Reductase 1 / Q000235:genetics*; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics
chemical_list: D000199:Actins; C576588:PABPN1 protein, human; D039122:Poly(A)-Binding Protein I; D012269:Ribosomal Proteins; C489210:Rpl13a protein, rat; C000606290:SOD1 protein, human; D035181:TATA-Box Binding Protein; D014446:Tyrosine 3-Monooxygenase; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D054481:Thioredoxin Reductase 1; C516126:Txnrd1 protein, rat; D007041:Hypoxanthine Phosphoribosyltransferase; D011163:Hydroxymethylbilane Synthase; D005966:Glucuronidase