Abstract
Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data.
Highlights
Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for quantifying gene transcription
Since the number of copies for the different expressed repetitive elements (ERE) in the mouse genome was unknown in Repbase, initially 16 EREs were selected based on a mean conservation rate of minimum 97% compared to the Repbase consensus sequence
The cornerstone of reliable RT-qPCR results is the use of an adequate number of validated and stably expressed reference genes for data normalization
Summary
Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for quantifying gene transcription. Commercial “endogenous control gene panels” for validation of RT-qPCR mouse reference genes are available These panels comprise the most commonly used reference genes in mouse research and these genes are considered to be generally stable under a wide variety of circumstances (e.g. across different tissues, mouse strains, experimental conditions, etc.). Our lab pioneered the use of expressed repetitive elements (ERE) as reference targets for RT-qPCR normalization in human and zebrafish samples[8,9,10,11]. In the zebrafish study, EREs were shown to outperform commonly used reference genes in a variety of experimental set-ups[8]. We compared the expression stability of mouse ERE reference targets and commonly used reference genes in six different experimental studies. We demonstrate that the murine EREs outperform the frequently used reference genes in a broad range of experimental set-ups
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