Importance of Suitable Reference Gene Selection for Quantitative Real-Time PCR: Special Reference to Mouse Myocardial Infarction Studies

BackgroundQuantitative real-time PCR (qPCR) is a widely used technique for gene expression analysis. Its reliability is highly dependent upon selection of the appropriate reference genes for accurate gene expression normalization. In this study, we investigated the expression stability of 10 commonly used reference genes in a mouse myocardial infarction model. Methods & ResultsThe expression stability of the 10 reference genes (Actb, B2m, Eef1a1, Gapdh, Hprt, Polr2a, Ppia, Rpl13a, Tbp, Tpt1) was analyzed using the geNorm software. Overall, the combination of Hprt, Rpl13a and Tpt1 was the most stable reference gene set in our experiments. Gapdh, Polr2a and Actb consistently showed the highest gene expression variability and the expression levels of Gapdh, Polr2a, Actb, B2m and Eef1a1 were found to be selectively up- or downregulated after myocardial infarction. We normalized the expression of Nppb and Vcam1, using different reference gene strategies and demonstrated that their induction after myocardial infarction was most clearly revealed with the optimal reference gene combination. However, the use of suboptimal reference gene combinations resulted in detrimental effects on gene expression levels and variability with a gradual loss of the expression differences and a significant reduction in statistical power. ConclusionsHprt, Rpl13a and Tpt1 are a set of stably expressed reference genes for accurate gene expression normalization in myocardial infarction studies in mice. We found that Gapdh, Polr2a and Actb display high expression variability in mouse myocardial infarction tissues and that loss of statistical power and increase in sample size are the evident consequences of choosing suboptimal combinations of reference genes. We furthermore caution against the use of Gapdh, Polr2a, Actb, B2m and Eef1a1 for gene expression normalization in myocardial infarction studies because of selective up- or downregulation after myocardial infarction, which could potentially lead to biased study outcomes.背景 定量实时聚合酶链反应（quantitative real-time PCR, qPCR）是一种广泛应用于基因表达分析的技术，其可靠性高度依赖于合适内参基因的筛选，以实现精准的基因表达标准化。本研究针对小鼠心肌梗死模型中的10种常用内参基因的表达稳定性展开了探究。 方法与结果 采用geNorm软件对这10种内参基因（Actb、B2m、Eef1a1、Gapdh、Hprt、Polr2a、Ppia、Rpl13a、Tbp、Tpt1）的表达稳定性进行分析。实验结果显示，Hprt、Rpl13a与Tpt1的组合为本研究中最稳定的内参基因集。Gapdh、Polr2a及Actb的基因表达变异度始终处于较高水平；而Gapdh、Polr2a、Actb、B2m及Eef1a1的表达水平在心肌梗死造模后呈现出选择性上调或下调趋势。本研究采用多种内参基因策略对Nppb与Vcam1的表达进行标准化，结果表明，使用最优内参基因组合可最清晰地展现二者在心肌梗死造模后的诱导表达特征。然而，使用次优内参基因组合会对基因表达水平及变异度产生不利影响，导致表达差异逐渐消失，同时统计效力显著降低。 结论 Hprt、Rpl13a与Tpt1是一组可用于小鼠心肌梗死研究中精准基因表达标准化的稳定表达内参基因。本研究发现，Gapdh、Polr2a及Actb在小鼠心肌梗死组织中表现出较高的表达变异度；选择次优内参基因组合会导致统计效力下降，且需增加样本量，这是显而易见的后果。此外，鉴于Gapdh、Polr2a、Actb、B2m及Eef1a1在心肌梗死造模后会出现选择性表达失调，本研究提醒研究者勿将其用于心肌梗死研究中的基因表达标准化，否则可能导致研究结果出现偏倚。