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: 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)

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

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)