Research article
Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies
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* Corresponding author: Kristin B Andersson k.b.andersson@medisin.uio.no
1 Institute for Experimental Medical Research, Oslo University Hospital Ullevål, Oslo, Norway
2 Department of Pharmacology, University of Oslo, Oslo, Norway
3 Center for Heart Failure Research, Faculty of Medicine, University of Oslo, Oslo, Norway
4 Department of Informatics, University of Oslo, Oslo, Norway
5 Current address: National Institute of Nutrition and Seafood Research, Bergen, Norway
BMC Molecular Biology 2010, 11:22 doi:10.1186/1471-2199-11-22
Published: 23 March 2010Abstract
Background
Quantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium.
Results
The abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions.
Conclusions
The most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.