Abstract
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.
Similar content being viewed by others
References
Burmester GR, Bijlsma JWJ, Cutolo M, McInnes IB. Managing rheumatic and musculoskeletal diseases—past, present and future. Nat Rev Rheumatol. 2017;13:443–8.
Kłak A, Raciborski F, Samel-Kowalik P. Social implications of rheumatic diseases. Reumatologia. 2016;54:73–8.
Hong J-I, Park IY, Kim HA. Understanding the molecular mechanisms underlying the pathogenesis of arthritis pain using animal models. Int J Mol Sci. 2020;21:533.
Choudhary N, Bhatt LK, Prabhavalkar KS. Experimental animal models for rheumatoid arthritis. Immunopharmacol Immunotoxicol. 2018;40:193–200.
McNamee K, Williams R, Seed M. Animal models of rheumatoid arthritis: How informative are they? Eur J Pharmacol. 2015;759:278–86.
Bessis N, Decker P, Assier E, Semerano L, Boissier M-C. Arthritis models: usefulness and interpretation. Semin Immunopathol. 2017;39:469–86.
Caplazi P, Baca M, Barck K, Carano RAD, DeVoss J, Lee WP, et al. Mouse models of rheumatoid arthritis. Vet Pathol. 2015;52:819–26.
Wang Y, Zhou C, Gao H, Li C, Li D, Liu P, et al. Therapeutic effect of Cryptotanshinone on experimental rheumatoid arthritis through downregulating p300 mediated-STAT3 acetylation. Biochem Pharmacol. 2017;138:119–29.
Hu Y, Li J, Qin L, Cheng W, Lai Y, Yue Y, et al. Study in treatment of collagen-induced arthritis in DBA/1 mice model by genistein. Curr Pharm Des. 2016;22:6975–81.
Al-Riyami L, Pineda MA, Rzepecka J, Huggan JK, Khalaf AI, Suckling CJ, et al. Designing anti-inflammatory drugs from parasitic worms: a synthetic small molecule analogue of the Acanthocheilonema viteae product ES-62 prevents development of collagen-induced arthritis. J Med Chem. 2013;56:9982–10002.
Maini RN, Elliott MJ, Brennan FM, Williams RO, Chu CQ, Paleolog E, et al. Monoclonal anti-TNF alpha antibody as a probe of pathogenesis and therapy of rheumatoid disease. Immunol Rev. 1995;144:195–223.
Miyoshi M, Liu S. Collagen-induced arthritis models. Methods Mol Biol Clifton NJ. 2018;1868:3–7.
Braem K, Carter S, Lories RJ. Spontaneous arthritis and ankylosis in male DBA/1 mice: further evidence for a role of behavioral factors in “stress-induced arthritis.” Biol Proced Online. 2012;14:10.
Alam J, Jantan I, Bukhari SNA. Rheumatoid arthritis: Recent advances on its etiology, role of cytokines and pharmacotherapy. Biomed Pharmacother Biomedecine Pharmacother. 2017;92:615–33.
González-Chávez SA, Quiñonez-Flores CM, Pacheco-Tena C. Molecular mechanisms of bone formation in spondyloarthritis. Jt Bone Spine Rev Rhum. 2016;83:394–400.
Xu Y, Zhu Q, Song J, Liu H, Miao Y, Yang F, et al. Regulatory effect of iguratimod on the balance of Th subsets and inhibition of inflammatory cytokines in patients with rheumatoid arthritis. Mediators Inflamm. 2015;2015:356040.
Sundberg E, Grundtman C, Af Klint E, Lindberg J, Ernestam S, Ulfgren A-K, et al. Systemic TNF blockade does not modulate synovial expression of the pro-inflammatory mediator HMGB1 in rheumatoid arthritis patients–a prospective clinical study. Arthritis Res Ther. 2008;10:R33.
Kim K-W, Cho M-L, Lee S-H, Oh H-J, Kang C-M, Ju JH, et al. Human rheumatoid synovial fibroblasts promote osteoclastogenic activity by activating RANKL via TLR-2 and TLR-4 activation. Immunol Lett. 2007;110:54–64.
Mallinson DJ, Dunbar DR, Ridha S, Sutton ER, De la Rosa O, Dalemans W, et al. Identification of potential plasma microRNA stratification biomarkers for response to allogeneic adipose-derived mesenchymal stem cells in rheumatoid arthritis. Stem Cells Transl Med. 2017;6:1202–6.
Guénin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot. 2009;60:487–93.
Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005;6:279–84.
Setiawan M, Jäger A, Konermann A. The stability of different housekeeping genes in human periodontal ligament cells under inflammatory conditions. Ann Anat. 2019;224:81–7.
Bamias G, Goukos D, Laoudi E, Balla IG, Siakavellas SI, Daikos GL, et al. Comparative study of candidate housekeeping genes for quantification of target gene messenger RNA expression by real-time PCR in patients with inflammatory bowel disease. Inflamm Bowel Dis. 2013;19:2840–7.
Brand DD, Latham KA, Rosloniec EF. Collagen-induced arthritis. Nat Protoc. 2007;2:1269–75.
De Spiegelaere W, Dern-Wieloch J, Weigel R, Schumacher V, Schorle H, Nettersheim D, et al. Reference gene validation for RT-qPCR, a note on different available software packages. PLoS ONE. 2015;10:e0122515.
Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis for real-time quantitative PCR data. Genome Biol. 2007;8:R19.
Pombo-Suarez M, Calaza M, Gomez-Reino JJ, Gonzalez A. Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage. BMC Mol Biol. 2008;9:17.
Li H, Chen C, Yao H, Li X, Yang N, Qiao J, et al. Identification of suitable reference genes for mRNA studies in bone marrow in a mouse model of hematopoietic stem cell transplantation. Transplant Proc. 2016;48:2826–32.
Qureshi MAA, Ahmed AS, Li J, Stark A, Eriksson P, Ahmed M. Validation of reference genes for mRNA quantification in adjuvant arthritis. Open J Rheumatol Autoimmune Dis. 2012;02:64–72.
Studer D, Lischer S, Jochum W, Ehrbar M, Zenobi-Wong M, Maniura-Weber K. Ribosomal protein l13a as a reference gene for human bone marrow-derived mesenchymal stromal cells during expansion, adipo-, chondro-, and osteogenesis. Tissue Eng Part C Methods. 2012;18:761–71.
Meng M, Liu H, Chen S, Zhao H, Gao X, Zhang J, et al. Methylation of H3K27 and H3K4 in key gene promoter regions of thymus in RA mice is involved in the abnormal development and differentiation of iNKT cells. Immunogenetics. 2019;71:489–99.
Bai L, Liu W, Guo P, Bai J, Liu Y, Hua Y, et al. Elevated levels of soluble Endothelial protein C receptor in rheumatoid arthritis and block the therapeutic effect of protein C in collagen-induced arthritis. Int Immunopharmacol. 2020;81:106255.
Dam TT, Hanaoka H, Nakajima T, Yamaguchi A, Okamura K, Chikuda H, et al. 64Cu-ATSM and 99mTc(CO)3-DCM20 potential in the early detection of rheumatoid arthritis. Mod Rheumatol. 2021;31:350–6.
Zhang Q, Li Q, Zhu J, Guo H, Zhai Q, Li B, et al. Comparison of therapeutic effects of different mesenchymal stem cells on rheumatoid arthritis in mice. PeerJ. 2019;7:e7023.
Song G, Lu Q, Fan H, Zhang X, Ge L, Tian R, et al. Inhibition of hexokinases holds potential as treatment strategy for rheumatoid arthritis. Arthritis Res Ther. 2019;21:87.
Liu R, Chen Y, Fu W, Wang S, Cui Y, Zhao X, et al. Fexofenadine inhibits TNF signaling through targeting to cytosolic phospholipase A2 and is therapeutic against inflammatory arthritis. Ann Rheum Dis. 2019;78:1524–35.
Li H, Cao X-Y, Dang W-Z, Jiang B, Zou J, Shen X-Y. Total Glucosides of Paeony protects against collagen-induced mouse arthritis via inhibiting follicular helper T cell differentiation. Phytomed Int J Phytother Phytopharm. 2019;65:153091.
Nho J-H, Kim A-H, Jung H-K, Lee M-J, Jang J-H, Yang B-D, et al. Water extract of acori graminei rhizoma attenuates features of rheumatoid arthritis in DBA/1 mice. Evid Based Complement Altern Med ECAM. 2019;2019:3637453.
Chen Y, Wang X, Yang M, Ruan W, Wei W, Gu D, et al. miR-145-5p increases osteoclast numbers in vitro and aggravates bone erosion in collagen-induced arthritis by targeting osteoprotegerin. Med Sci Monit Int Med J Exp Clin Res. 2018;24:5292–300.
Luo X, Chen Y, Lv G, Zhou Z, Chen J, Mo X, et al. Adenovirus-mediated small interfering RNA targeting TAK1 ameliorates joint inflammation with collagen-induced arthritis in mice. Inflammation. 2017;40:894–903.
Gonzalo-Gil E, Pérez-Lorenzo MJ, Galindo M, de la Díaz GR, López-Millán B, Bueno C, et al. Human embryonic stem cell-derived mesenchymal stromal cells ameliorate collagen-induced arthritis by inducing host-derived indoleamine 2,3 dioxygenase. Arthritis Res Ther. 2016;18:77.
Zhou Y-R, Zhao Y, Bao B-H, Li J-X. SND-117, a sinomenine bivalent alleviates type II collagen-induced arthritis in mice. Int Immunopharmacol. 2015;26:423–31.
Zhu L, Zhu L. Sophocarpine suppress inflammatory response in human fibroblast-like synoviocytes and in mice with collagen-induced arthritis. Eur Cytokine Netw. 2017;28:120–6.
Sun X, Wang Y, Zhang M, Wang Q. Intraarticular injection of dexamethasone promotes bone erosion in collagen-induced arthritis in mice through up-regulation of RANKL expression. Inflammopharmacology. 2019;27:503–9.
Li S, Xiang C, Wei X, Sun X, Li R, Li P, et al. Early supplemental α2-macroglobulin attenuates cartilage and bone damage by inhibiting inflammation in collagen II-induced arthritis model. Int J Rheum Dis. 2019;22:654–65.
Oh BR, Suh D-H, Bae D, Ha N, Choi YI, Yoo HJ, et al. Therapeutic effect of a novel histone deacetylase 6 inhibitor, CKD-L, on collagen-induced arthritis in vivo and regulatory T cells in rheumatoid arthritis in vitro. Arthritis Res Ther. 2017;19:154.
Al-Sabah A, Stadnik P, Gilbert SJ, Duance VC, Blain EJ. Importance of reference gene selection for articular cartilage mechanobiology studies. Osteoarthritis Cartilage. 2016;24:719–30.
Nazet U, Schröder A, Grässel S, Muschter D, Proff P, Kirschneck C. Housekeeping gene validation for RT-qPCR studies on synovial fibroblasts derived from healthy and osteoarthritic patients with focus on mechanical loading. PLoS ONE. 2019;14:e0225790.
Schildberg T, Rauh J, Bretschneider H, Stiehler M. Identification of suitable reference genes in bone marrow stromal cells from osteoarthritic donors. Stem Cell Res. 2013;11:1288–98.
Watanabe H, Ishii H, Takahashi K, Takai S, Ozawa H. Suitable reference gene selection for gene expression studies in knee osteoarthritis synovium using quantitative PCR analysis. Connect Tissue Res. 2018;59:356–68.
Leal MF, Arliani GG, Astur DC, Franciozi CE, Debieux P, Andreoli CV, et al. Comprehensive selection of reference genes for expression studies in meniscus injury using quantitative real-time PCR. Gene. 2016;584:60–8.
Jiang C, Meng L, Zhu W, Shahzad M, Yang X, Lu S. Housekeeping gene stability in pristane-induced arthritis and antigen-induced pulmonary inflammation of rats. Inflamm Res. 2009;58:601–9.
Montero-Melendez T, Perretti M. Gapdh gene expression is modulated by inflammatory arthritis and is not suitable for qPCR normalization. Inflammation. 2014;37:1059–69.
Hanafy S, Jamali F. Adjuvant arthritis influences expression of housekeeping genes. Inflamm Res. 2011;60:521–3.
Funding
The authors declare no funding regarding the publication of this article.
Author information
Authors and Affiliations
Contributions
Conceptualization: SAG-C; Methodology: SAG-C; Software: CMQ-F, SML-L; Validation: CMQ-F, SML-L; Formal analysis: SAG-C, CMQ-F, SML-L, CP-T; Investigation: SAG-C, CMQ-F, SML-L, PMM-M, SA-J; Resources: CP-T; Data curation: SAG-C, CMQ-F, SML-L, CP-T; Writing—original draft preparation: SAG-C, CMQ-F, SML-L; Writing—review and editing: CP-T, PMM-M, SA-J; Visualization: SAG-C; Supervision: CP-T; Project administration: SAG-C; Funding acquisition: SAG-C, CP-T.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests regarding the publication of this article.
Additional information
Responsible Editor: John Di Battista.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Quiñonez-Flores, C.M., López-Loeza, S.M., Pacheco-Tena, C. et al. Stability of housekeeping genes in inflamed joints of spontaneous and collagen-induced arthritis in DBA/1 mice. Inflamm. Res. 70, 619–632 (2021). https://doi.org/10.1007/s00011-021-01453-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00011-021-01453-2