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.
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Authors | Nicoletta Vesentini, Cristina Barsanti, Alessandro Martino, Claudia Kusmic, Andrea Ripoli, AnnaMaria Rossi, Antonio L'Abbate |
Journal | BMC research notes
(BMC Res Notes)
Vol. 5
Pg. 124
(Feb 29 2012)
ISSN: 1756-0500 [Electronic] England |
PMID | 22377061
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Actins
- PABPN1 protein, human
- Poly(A)-Binding Protein I
- Ribosomal Proteins
- Rpl13a protein, rat
- SOD1 protein, human
- TATA-Box Binding Protein
- Tyrosine 3-Monooxygenase
- Sod1 protein, rat
- Superoxide Dismutase
- Superoxide Dismutase-1
- Glyceraldehyde-3-Phosphate Dehydrogenases
- Thioredoxin Reductase 1
- Txnrd1 protein, rat
- Hypoxanthine Phosphoribosyltransferase
- Hydroxymethylbilane Synthase
- Glucuronidase
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Topics |
- Actins
(genetics)
- Animals
- Disease Models, Animal
- Gene Expression Profiling
(standards)
- Glucuronidase
(genetics)
- Glyceraldehyde-3-Phosphate Dehydrogenases
(genetics)
- Hydroxymethylbilane Synthase
(genetics)
- Hypoxanthine Phosphoribosyltransferase
(genetics)
- Male
- Myocardial Reperfusion Injury
(genetics)
- Myocardium
(metabolism)
- Poly(A)-Binding Protein I
(genetics)
- Rats
- Rats, Wistar
- Reference Standards
- Reverse Transcriptase Polymerase Chain Reaction
- Ribosomal Proteins
(genetics)
- Superoxide Dismutase
(genetics)
- Superoxide Dismutase-1
- TATA-Box Binding Protein
(genetics)
- Thioredoxin Reductase 1
(genetics)
- Time Factors
- Tyrosine 3-Monooxygenase
(genetics)
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