ArticlePloS one2011
Importance of suitable reference gene selection for quantitative real-time PCR: special reference to mouse myocardial infarction studies.
Article in PloS one, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 41 papers.
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41 citing papers in PubMed, 78 citations in OpenAlex.
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- Selection of species specific panel of reference genes in peripheral blood mononuclear cells of native livestock species adapted to trans-Himalayan region of Leh-Ladakh.Scientific reports · 2022Article
- Housekeeping Proteins Exhibit a High Level of Expression Variability Within Control Group and Between Ischemic Human Heart Biopsies.Journal of the American Heart Association · 2022Article
- Normalization strategy for selection of reference genes for RT-qPCR analysis in left ventricles of failing human hearts.BMC cardiovascular disorders · 2022Article
- Identification of reference genes for gene expression studies among different developmental stages of murine hearts.BMC developmental biology · 2021Article
- Single-cell transcriptomics following ischemic injury identifies a role for B2M in cardiac repair.Communications biology · 2021Article
- Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis ofFrontiers in microbiology · 2021Article
- Evaluation of qPCR reference genes in GH-overexpressing transgenic zebrafish (Danio rerio).Scientific reports · 2020Article
- Exposure to graphene oxide sheets alters the expression of reference genes used for real-time RT-qPCR normalization.Scientific reports · 2019Article
- Validation of Reference Genes for Gene Expression Normalization in RAW264.7 Cells under Different Conditions.BioMed research international · 2019Article
- What's the norm in normalization? A frightening note on the use of RT-qPCR in the livestock science.Gene · 2019Article
- Arginine reprogramming in ADPKD results in arginine-dependent cystogenesis.American journal of physiology. Renal physiology · 2018Article
- Identification of reference genes for quantitative PCR analyses in developing mouse gonads.The Journal of veterinary medical science · 2018Article
- Increases in plasma corin levels following experimental myocardial infarction reflect the severity of ischemic injury.PloS one · 2018Article
- A Tunable, Three-Dimensional In Vitro Culture Model of Growth Plate Cartilage Using Alginate Hydrogel Scaffolds.Tissue engineering. Part A · 2018Article
- Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition.PloS one · 2018Article
- Identification of TMEM208 and PQLC2 as reference genes for normalizing mRNA expression in colorectal cancer treated with aspirin.Oncotarget · 2017Article
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Authors and funding
4 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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