Identification of reference genes for gene expression studies among different developmental stages of murine hearts

Jie Ren,Ningning Zhang,Xiangjie Li,Xiaogang Sun,Jiangping Song

doi:10.1186/s12861-021-00244-6

Jie Ren, Ningning Zhang + Show 3 more

Open Access

https://doi.org/10.1186/s12861-021-00244-6

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Abstract

BackgroundReal-time quantitative polymerase chain reaction (RT-qPCR) is a widely-used standard assay for assessing gene expression. RT-qPCR data requires reference genes for normalization to make the results comparable. Therefore, the selected reference gene should be highly stable in its expression throughout the experimental datasets. So far, reports about the optimal set of reference genes in murine left ventricle (LV) across embryonic and postnatal stages are few. The objective of our research was to identify the appropriate reference genes in murine LV among different developmental stages.MethodsWe investigated the gene expression profiles of 21 widely used housekeeping genes in murine LV from 7 different developmental stages (almost throughout the whole period of the mouse lifespan). The stabilities of the potential reference genes were evaluated by five methods: GeNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder.ResultsWe proposed a set of reliable reference genes for normalization of RT-qPCR experimental data in different conditions. Furthermore, our results showed that 6 genes (18S, Hmbs, Ubc, Psmb4, Tfrc and Actb) are not recommended to be used as reference genes in murine LV development studies. The data also suggested that the Rplp0 gene might serve as an optimal reference gene in gene expression analysis.ConclusionsOur study investigated the expression stability of the commonly used reference genes in process of LV development and maturation. We proposed a set of optimal reference genes that are suitable for accurate normalization of RT-qPCR data in specific conditions. Our findings may be helpful in future studies for investigating the gene expression patterns and mechanism of mammalian heart development.

Highlights

Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely-used standard assay for assessing gene expression
We found that some genes have more stable expression across different developmental stages, than others, e.g., Pgk1, Tfrc, actin beta (Actb) (Additional file 2: Table S1)
We propose a set of optimal reference genes under different conditions and suggest Rplp0 could serve as a stable reference gene of left ventricle (LV) tissue across different developmental stages

Summary

Introduction

Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely-used standard assay for assessing gene expression. RT-qPCR data requires reference genes for normalization to make the results comparable. The selected reference gene should be highly stable in its expression throughout the experimental datasets. The objective of our research was to identify the appropriate reference genes in murine LV among different developmental stages. Real-time quantitative polymerase chain reaction (RTqPCR) is still the standard assay used for quantification of gene expression with high sensitivity and accuracy [11]. RT-qPCR data needs reference genes for normalization to make the results comparable [12]. Selection of appropriate reference gene is important in the design of a RT-qPCR experiment [13]. It is critical to identify suitable reference genes with relatively stable expression in the specific context

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