Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli

Abstract

Tendon cells (TCs) are important for homeostatic maintenance in the healthy tendon and to promote tissue healing after injury. Further, resident and rare populations of tendon stem/progenitor cells, located at various sites within the tendon, contribute to tendon recovery by differentiating into repairing TCs. Gene expression analysis, through quantitative reverse-transcription polymerase chain reaction (qRT-PCR), constitutes a useful tool to study cellular responses, including the transition from initial inflammation to healing processes. A critical step required for data normalization is the choice of reliable reference genes (RGs), a process highly underestimated in tendon biology. In this study, the suitability of five commonly used RGs (ACTB, B2M, GAPDH, HPRT1, and RPLP0) was evaluated using TCs samples cultured in both standard and progenitor-enriching conditions, as well as under either inflammatory (IFNγ + TNFα) or pro-fibrotic/healing (CTGF) stimulation. The stability of the candidate RGs was computationally determined using NormFinder, geNorm, BestKeeper, and DeltaCt applets. Overall, ACTB resulted as the most stable RG on the basis of the integration of each gene weight, whereas B2M and RPLP0 performed poorly. To further validate ACTB’s optimal performance, we evaluated the expression of ICAM1, coding for an immune-related cell surface glycoprotein, and COL1A1, encoding collagen type I that is the main component of the tendon extracellular matrix (ECM), both known to be modulated by inflammation. The expression of both genes was heavily affected by the RGs used. Consequently, when analyzing gene expression in tendon-derived cells subjected to various stimulatory protocols, the use of a suitable RG should be considered carefully. On the basis of our results, ACTB can be reliably used when analyzing different TC types exposed to pathological conditions.