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

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 rep...

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Autores principales: Ragni, Enrico, Perucca Orfei, Carlotta, Bowles, Annie C., de Girolamo, Laura, Correa, Diego
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830081/
https://www.ncbi.nlm.nih.gov/pubmed/31581587
http://dx.doi.org/10.3390/cells8101188
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author Ragni, Enrico
Perucca Orfei, Carlotta
Bowles, Annie C.
de Girolamo, Laura
Correa, Diego
author_facet Ragni, Enrico
Perucca Orfei, Carlotta
Bowles, Annie C.
de Girolamo, Laura
Correa, Diego
author_sort Ragni, Enrico
collection PubMed
description 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.
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spelling pubmed-68300812019-11-18 Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli Ragni, Enrico Perucca Orfei, Carlotta Bowles, Annie C. de Girolamo, Laura Correa, Diego Cells Article 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. MDPI 2019-10-01 /pmc/articles/PMC6830081/ /pubmed/31581587 http://dx.doi.org/10.3390/cells8101188 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ragni, Enrico
Perucca Orfei, Carlotta
Bowles, Annie C.
de Girolamo, Laura
Correa, Diego
Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli
title Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli
title_full Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli
title_fullStr Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli
title_full_unstemmed Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli
title_short Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli
title_sort reliable reference genes for gene expression assessment in tendon-derived cells under inflammatory and pro-fibrotic/healing stimuli
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830081/
https://www.ncbi.nlm.nih.gov/pubmed/31581587
http://dx.doi.org/10.3390/cells8101188
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