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Gapdh Gene Expression Is Modulated by Inflammatory Arthritis and Is not Suitable for qPCR Normalization

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Abstract

Gene expression studies are fundamental for the understanding of complex diseases, providing new insights into the pathogenic process and new tools for diagnostic and patient stratification. Gene profiling studies by real-time PCR require the use of reference genes for normalization and an appropriate validation is essential for accurate results. We performed a comprehensive assessment of six common housekeeping genes in the K/BxN serum-induced arthritis model in mice. Classical statistics and NormFinder analyses pointed out Gapdh as the less stable and therefore unsuitable as a reference control. Gapdh was considerably down-regulated in arthritic joints and therefore produced an overestimation of transcriptional changes. Hptr, B2m, and Rpl13a showed the most constant expression. Collectively our data advise against the use of Gapdh in gene expression studies in the acute phase of the K/BxN model and adds a cautionary note on the need to validate the reference genes for reliable, comparable, and reproducible results.

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Abbreviations

C T :

Threshold cycle

qPCR:

Quantitative polymerase chain reaction

RA:

Rheumatoid arthritis

RT:

Reverse transcription

WT:

Wild type

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ACKNOWLEDGMENTS

Funded by Medical Research Council, MR/K013068/1.

Conflict of Interest

Authors declare no competing interests.

Authors’ Contributions

TMM: designed the study, performed and analyzed experiments, and wrote manuscript.

MP: participated in the design of the study, helped to draft the manuscript, and provided funding.

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Authors and Affiliations

  1. The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, UK

    Trinidad Montero-Melendez & Mauro Perretti

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  1. Trinidad Montero-Melendez
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Correspondence to Trinidad Montero-Melendez.

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Montero-Melendez, T., Perretti, M. Gapdh Gene Expression Is Modulated by Inflammatory Arthritis and Is not Suitable for qPCR Normalization. Inflammation 37, 1059–1069 (2014). https://doi.org/10.1007/s10753-014-9829-x

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