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Normalization of Reverse Transcription Quantitative PCR Data During Ageing in Distinct Cerebral Structures

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Abstract

Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a routine method in many laboratories. Normalization of data from experimental conditions is critical for data processing and is usually achieved by the use of a single reference gene. Nevertheless, as pointed by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, several reference genes should be used for reliable normalization. Ageing is a physiological process that results in a decline of many expressed genes. Reliable normalization of RT-qPCR data becomes crucial when studying ageing. Here, we propose a RT-qPCR study from four mouse brain regions (cortex, hippocampus, striatum and cerebellum) at different ages (from 8 weeks to 22 months) in which we studied the expression of nine commonly used reference genes. With the use of two different algorithms, we found that all brain structures need at least two genes for a good normalization step. We propose specific pairs of gene for efficient data normalization in the four brain regions studied. These results underline the importance of reliable reference genes for specific brain regions in ageing.

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Acknowledgments

We would like to thank the Basse-Normandie region for funding Bruckert G and Dr Roussel BD and its credits for the laboratory.

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Authors and Affiliations

  1. INSERM, INSERM U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, GIP CYCERON, University Caen Basse Normandie, boulevard Becquerel, 14074, Caen, France

    G. Bruckert, D. Vivien, F. Docagne & B. D. Roussel

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  1. G. Bruckert
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  2. D. Vivien
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  3. F. Docagne
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  4. B. D. Roussel
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Corresponding author

Correspondence to B. D. Roussel.

Additional information

Neurodegenerative diseases represent a real challenge for our societies as their frequencies increase with ageing of the population. These diseases are often associated to gene regulation disorders that make their study useful for the comprehension of the disease. Currently, quantitative PCR is the most efficient tool for this kind of studies because it allows a fast and accurate measurement of gene expression levels. However, the reliability of RT-qPCR is influenced by the selection of reference genes, whose expression must be stable in the experimental conditions studied. However, many studies use “common” reference genes such as GAPDH or actin-beta, which have been reported to be unstable in certain conditions. Moreover, Sieber and collaborators have demonstrated the importance of using more than one reference gene to strengthen the exactness of measurements when studying ageing or brain insults such as ischaemia. However, the authors focused on the whole brain instead of studying several structures separately.

By using different algorithms, we identified specific combinations of housekeeping genes in the cortex, the hippocampus, the striatum and the cerebellum, allowing a reliable measurement of gene expression during a longitudinal study in RT-qPCR.

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Supplementary table 1

An intragroup analysis was performed using the Mann and Whitney non-parametric test (Grap Prism software) on the raw data for each genes studied in the cortex, hippocampus, striatum and cerebellum. *: p < 0.05; **: p < 0.01; ***: p < 0.001. (DOCX 23 kb)

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Bruckert, G., Vivien, D., Docagne, F. et al. Normalization of Reverse Transcription Quantitative PCR Data During Ageing in Distinct Cerebral Structures. Mol Neurobiol 53, 1540–1550 (2016). https://doi.org/10.1007/s12035-015-9114-5

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  • DOI: https://doi.org/10.1007/s12035-015-9114-5

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