Gapdh Gene Expression Is Modulated by Inflammatory Arthritis and Is not Suitable for qPCR Normalization
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Gapdh Gene Expression Is Modulated by Inflammatory Arthritis and Is not Suitable for qPCR Normalization Trinidad Montero-Melendez1,2 and Mauro Perretti1
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. KEY WORDS: arthritis; gene expression; inflammation; K/BxN serum-induced arthritis; real-time PCR.
INTRODUCTION Rheumatoid arthritis (RA) is a disabling and chronic inflammatory disease mainly affecting the joints. The aetiology is not completely understood and substantial efforts are constantly made by researchers for a better understanding of the initiation and progression of the disease, the variability observed in the response to therapy and in the identification of disease subtypes that could aid in the development of personalized therapeutic approaches [1–4]. Gene expression profiling is increasingly important in the study of RA as well as in many fields of biological research. The identification of gene expression patterns could aid for example in the development of new tools for diagnosis or patient stratification and might provide new 1
The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, UK 2 To whom correspondence should be addressed at The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, UK. E-mail: [email protected] ABBREVIATIONS: CT, Threshold cycle; qPCR, Quantitative polymerase chain reaction; RA, Rheumatoid arthritis; RT, Reverse transcription; WT, Wild type
insights into molecular mechanisms associated with the disease [5, 6]. The two main methods for measuring gene expression are microarrays, which allow the study of thousands of genes simultaneously, and quantitative real-time PCR (qPCR), which is the most sensitive method to characterize gene expression. There are two main approaches for the quantification of gene expression by qPCR: relative quantification, in which the transcripts levels are compared with a cont
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