Stability of housekeeping genes in inflamed joints of spontaneous and collagen-induced arthritis in DBA/1 mice

Background DBA/1 mice arthritis models have contributed to our understanding of human rheumatoid arthritis (RA) and spondyloarthritis (SpA) pathogenesis, as well as the exploration of therapeutic targets for treatment. Quantitative polymerase chain reaction (qPCR) is an indispensable tool in molecular research, which requires reference gene validation to obtain consistent and reliable results. Objective To determine the stability of candidate reference genes for qPCR in the joint of collagen-induced arthritis (CIA) and spontaneous arthritis (SpAD) DBA/1 mice. Methods The expression of eleven commonly used reference genes ( ACTB, B2M, EF1a, GAPDH, HMBS, HPRT, PPIB, RPL13A, SDHA, TBP, and YWHAZ ) was assessed by qPCR and the data were compared using delta-Ct methods and the geNorm, NormFinder, and RefFinder software packages. Genes identified as stable in each model were used for the quantification of inflammatory cytokines Results The gene stabilities differed between the two arthritis models in the DBA/1 mice. EF1a and RPL13A were the best reference genes for SpAD, while RPL13A and TBP were the best for the CIA. These genes allowed the data normalization for the quantification of the inflammatory cytokines in both models; these results showed an increase in the expression of IL-1B, IL-12B, IL-17A, and IL-6 in the inflamed joints. The use of different primer sequences for the same reference gene resulted in different relative quantification values. Conclusion This study demonstrates that commonly used reference genes may not be suitable for arthritic tissues from DBA/1 mice, and strengthening the principle that meticulous validation of reference genes is essential before each experiment to obtain valid and reproducible qPCR data for analysis or interpretation.