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.

中文翻译：

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DBA/1 小鼠自发性关节炎和胶原诱导性关节炎发炎关节中管家基因的稳定性

 背景

DBA/1小鼠关节炎模型有助于我们了解人类类风湿关节炎（RA）和脊柱关节炎（SpA）发病机制，以及探索治疗靶点。定量聚合酶链反应（qPCR）是分子研究中不可或缺的工具，需要参考基因验证才能获得一致可靠的结果。

 客观的

确定 qPCR 候选参考基因在胶原诱导性关节炎 (CIA) 和自发性关节炎 (SpAD) DBA/1 小鼠关节中的稳定性。

 方法

通过 qPCR 评估 11 个常用内参基因（ ACTB、B2M、EF1a、GAPDH、HMBS、HPRT、PPIB、RPL13A、SDHA、TBP 和YWHAZ ）的表达，并使用 delta-Ct 方法和 geNorm 比较数据， NormFinder 和 RefFinder 软件包。在每个模型中被鉴定为稳定的基因被用于炎症细胞因子的定量

 结果

DBA/1 小鼠的两种关节炎模型的基因稳定性不同。 EF1a和RPL13A是SpAD的最佳参考基因，而RPL13A和TBP是CIA的最佳参考基因。这些基因允许对两种模型中炎症细胞因子的量化进行数据标准化；这些结果表明发炎关节中 IL-1B、IL-12B、IL-17A 和 IL-6 的表达增加。对同一参考基因使用不同的引物序列会导致不同的相对定量值。

 结论

这项研究表明，常用的参考基因可能不适用于 DBA/1 小鼠的关节炎组织，并强调了在每次实验之前对参考基因进行细致验证至关重要的原则，以获得有效且可重复的 qPCR 数据进行分析或解释。