Comparative Biomedical Sciences, Royal Veterinary College, London, United Kingdom.
Clinical Sciences and Services, Royal Veterinary College, Hatfield, United Kingdom.
PLoS One. 2024 Aug 27;19(8):e0306678. doi: 10.1371/journal.pone.0306678. eCollection 2024.
Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tools for investigating tendon injury, allowing assessment of repair processes and pathological responses such as fibrosis, and permitting evaluation of therapeutic pharmacological interventions. Quantitative polymerase chain reaction (qPCR) is a commonly used approach for such studies, but data obtained by this method must be normalised to reference genes: genes known to be stably expressed between the experimental conditions investigated. Establishing suitable tendon injury reference genes is thus essential. Accordingly we investigated mRNA expression stability in a rat model of tendon injury, comparing both injured and uninjured tendons, and the effects of rapamycin treatment, at 1 and 3 weeks post injury. We used 11 candidate genes (18S, ACTB, AP3D1, B2M, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13a, SDHA, UBC) and assessed stability via four complementary algorithms (Bestkeeper, deltaCt, geNorm, Normfinder). Our results suggests that ACTB, CSNK2A2, HPRT1 and PAK1IP1 are all stably expressed in tendon, regardless of injury or drug treatment: any three of these would serve as universally suitable reference gene panel for normalizing qPCR expression data in the rat tendon injury model. We also reveal 18S, UBC, GAPDH, and SDHA as consistently poor scoring candidates (with the latter two exhibiting rapamycin- and injury-associated changes, respectively): these genes should be avoided.
肌腱是人体主要的承重组织之一；承受着巨大的峰值应力，因此容易受伤。细胞对肌腱损伤的反应很复杂，涉及炎症和修复成分，后者既利用常驻细胞群体，也利用募集的外源性细胞群体。基因表达分析是研究肌腱损伤的有价值的工具，可以评估修复过程和纤维化等病理反应，并允许评估治疗性药理干预。定量聚合酶链反应 (qPCR) 是此类研究中常用的方法，但该方法获得的数据必须通过参考基因进行归一化：即在研究的实验条件下已知稳定表达的基因。因此，建立合适的肌腱损伤参考基因是必不可少的。相应地，我们在肌腱损伤的大鼠模型中研究了 mRNA 表达稳定性，比较了受伤和未受伤的肌腱，以及雷帕霉素治疗在损伤后 1 周和 3 周的影响。我们使用了 11 个候选基因（18S、ACTB、AP3D1、B2M、CSNK2A2、GAPDH、HPRT1、PAK1IP1、RPL13a、SDHA、UBC），并通过四种互补算法（Bestkeeper、deltaCt、geNorm、Normfinder）评估了稳定性。我们的结果表明，ACTB、CSNK2A2、HPRT1 和 PAK1IP1 在肌腱中均稳定表达，无论损伤或药物治疗如何：这四个基因中的任何三个都可以作为普遍适用的参考基因面板，用于标准化大鼠肌腱损伤模型中的 qPCR 表达数据。我们还揭示了 18S、UBC、GAPDH 和 SDHA 作为始终表现不佳的候选基因（后两者分别表现出雷帕霉素和损伤相关的变化）：应避免使用这些基因。
