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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.

Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies