Selection of reference genes in different myocardial regions of an in vivo ischemia/reperfusion rat model for normalization of antioxidant gene expression

BMC Res Notes. 2012 Feb 29;5:124. doi: 10.1186/1756-0500-5-124.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • 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