Gapdh gene expression is modulated by inflammatory arthritis and is not suitable for qPCR normalization.
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.
The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, UK, t.monteromelendez@qmul.ac.uk.
Trinidad
T
Montero-Melendez
Mauro
M
Perretti
D000818:Animals; D001168:Arthritis / Q000188:drug therapy*; D004195:Disease Models, Animal; D015870:Gene Expression; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics / Q000378:metabolism*; D007249:Inflammation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*
D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)