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

<p>Abstract</p> <p>Background</p> <p>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 <it>Gapdh </it>mRNA and <it>18S </it>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.</p> <p>Results</p> <p>The abundance of <it>Arbp</it>, <it>Rpl32</it>, <it>Rpl4</it>, <it>Tbp</it>, <it>Polr2a</it>, <it>Hprt1</it>, <it>Pgk1</it>, <it>Ppia </it>and <it>Gapdh </it>mRNA and <it>18S </it>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.</p> <p>Conclusions</p> <p>The most stable reference genes were <it>Rpl32</it>, <it>Gapdh </it>and <it>Polr2a </it>in mouse post-infarction heart failure, <it>Polr2a</it>, <it>Rpl32 </it>and <it>Tbp </it>in rat post-infarction heart failure and <it>Rpl32 </it>and <it>Pgk1 </it>in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was <it>Rpl32 </it>and <it>Polr2a</it>. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with <it>Rpl4 </it>as was most stable among the tested genes.</p