Institute for Experimental Medical Research, Oslo University Hospital Ullevål, Oslo, Norway.
BMC Mol Biol. 2010 Mar 23;11:22. doi: 10.1186/1471-2199-11-22.
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.
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.
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.
实时定量 RT-PCR（RT-qPCR）是一种高度敏感的 mRNA 定量方法，但需要所选参考基因（s）的不变表达。在病理性心肌中，除了常用的 Gapdh mRNA 和 18S 核糖体 RNA 之外，关于其他合适的参考基因的信息有限。我们的目的是评估和确定人类衰竭心肌、大鼠和小鼠心肌梗死后心力衰竭以及胎鼠和新生鼠心肌发育阶段的合适参考基因。
通过 RT-qPCR 定量测定心肌样本中 Arbp、Rpl32、Rpl4、Tbp、Polr2a、Hprt1、Pgk1、Ppia 和 Gapdh mRNA 和 18S 核糖体 RNA 的丰度。通过 geNorm 和 Normfinder 算法以及方差分量分析方法评估这些转录本的表达变异性。生物变异性比重复逆转录或 PCR 反应对样品变异性的贡献更大。
在小鼠梗死后心力衰竭中，最稳定的参考基因是 Rpl32、Gapdh 和 Polr2a；在大鼠梗死后心力衰竭中，最稳定的参考基因是 Polr2a、Rpl32 和 Tbp；在人类心力衰竭（缺血性疾病和心肌病）中，最稳定的参考基因是 Rpl32 和 Pgk1。在所有三个物种中，最稳定的参考基因是 Rpl32 和 Polr2a。在大鼠心肌中，所有测试的参考基因都显示出与发育阶段的显著变化，其中 Rpl4 是测试基因中最稳定的。
