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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MDPI AG
2019-10-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/8/10/1188 |
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| author | Enrico Ragni Carlotta Perucca Orfei Annie C. Bowles Laura de Girolamo Diego Correa |
| author_facet | Enrico Ragni Carlotta Perucca Orfei Annie C. Bowles Laura de Girolamo Diego Correa |
| author_sort | Enrico Ragni |
| collection | DOAJ |
| 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 (<i>ACTB</i>, <i>B2M</i>, <i>GAPDH</i>, <i>HPRT1</i>, and <i>RPLP0</i>) 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, <i>ACTB</i> resulted as the most stable RG on the basis of the integration of each gene weight, whereas <i>B2M</i> and <i>RPLP0</i> performed poorly. To further validate <i>ACTB</i>’s optimal performance, we evaluated the expression of <i>ICAM1</i>, coding for an immune-related cell surface glycoprotein, and <i>COL1A1</i>, 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, <i>ACTB</i> can be reliably used when analyzing different TC types exposed to pathological conditions. |
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| language | English |
| last_indexed | 2024-03-12T08:19:31Z |
| publishDate | 2019-10-01 |
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| spelling | doaj.art-23de63dc42fc41708c8a37bdbb313ee32023-09-02T18:34:07ZengMDPI AGCells2073-44092019-10-01810118810.3390/cells8101188cells8101188Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing StimuliEnrico Ragni0Carlotta Perucca Orfei1Annie C. Bowles2Laura de Girolamo3Diego Correa4IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all’Ortopedia, Via R. Galeazzi 4, I-20161 Milano, ItalyIRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all’Ortopedia, Via R. Galeazzi 4, I-20161 Milano, ItalyDepartment of Orthopedics, UHealth Sports Medicine Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USAIRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all’Ortopedia, Via R. Galeazzi 4, I-20161 Milano, ItalyDepartment of Orthopedics, UHealth Sports Medicine Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USATendon 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 (<i>ACTB</i>, <i>B2M</i>, <i>GAPDH</i>, <i>HPRT1</i>, and <i>RPLP0</i>) 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, <i>ACTB</i> resulted as the most stable RG on the basis of the integration of each gene weight, whereas <i>B2M</i> and <i>RPLP0</i> performed poorly. To further validate <i>ACTB</i>’s optimal performance, we evaluated the expression of <i>ICAM1</i>, coding for an immune-related cell surface glycoprotein, and <i>COL1A1</i>, 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, <i>ACTB</i> can be reliably used when analyzing different TC types exposed to pathological conditions.https://www.mdpi.com/2073-4409/8/10/1188tendontenocyteprogenitor cellinflammationreference geneqrt-pcr |
| spellingShingle | Enrico Ragni Carlotta Perucca Orfei Annie C. Bowles Laura de Girolamo Diego Correa Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli Cells tendon tenocyte progenitor cell inflammation reference gene qrt-pcr |
| 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 | tendon tenocyte progenitor cell inflammation reference gene qrt-pcr |
| url | https://www.mdpi.com/2073-4409/8/10/1188 |
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