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International journal of molecular sciences2024; 25(4); 2292; doi: 10.3390/ijms25042292

Identification of the Reference Genes for Relative qRT-PCR Assay in Two Experimental Models of Rabbit and Horse Subcutaneous ASCs.

Abstract: Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore, in our study, we comprehensively assessed and compared the suitability of some widely used HKGs, employing RefFinder and NormFinder, two extensively acknowledged algorithms for robust data interpretation. The rabbit and horse ASCs were obtained from subcutaneous stromal vascular fraction. ASCs were induced into tri-lineage differentiation, followed by the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) treatment of the adipose-differentiated rabbit ASCs, while horse experimental groups were formed based on adipogenic, osteogenic, and chondrogenic differentiation. At the end of the experiment, the total mRNA was obtained and used for the gene expression evaluation of the observed factors. According to our findings, glyceraldehyde 3-phosphate dehydrogenase was identified as the most appropriate endogenous control gene for rabbit ASCs, while hypoxanthine phosphoribosyltransferase was deemed most suitable for horse ASCs. The obtained results underscore that these housekeeping genes exhibit robust stability across diverse experimental conditions, remaining unaltered by the treatments. In conclusion, the current research can serve as a valuable baseline reference for experiments evaluating gene expression in rabbit and horse ASCs. It highlights the critical consideration of housekeeping gene abundance and stability in qPCR experiments, emphasizing the need for an individualized approach tailored to the specific requirements of the study.
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Publication Date: 2024-02-14 PubMed ID: 38396967PubMed Central: PMC10889259DOI: 10.3390/ijms25042292Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study identifies the most stable reference genes for normalizing qRT-PCR data in adipose-derived stem cells (ASCs) from rabbits and horses under various experimental conditions.
  • The researchers evaluated common housekeeping genes to ensure accurate gene expression analysis in these specific stem cell models.

Introduction and Importance of Reference Genes

  • qRT-PCR is a widely-used technique for measuring gene expression levels, but data accuracy depends heavily on normalization.
  • Normalization is done using reference genes, typically housekeeping genes (HKGs), which should have consistent expression across different samples and conditions.
  • Choosing inappropriate reference genes can lead to misleading or incorrect results.
  • There is limited information about the best reference genes for adipose-derived stem cells from rabbits and horses, which are increasingly used in regenerative medicine research.

Experimental Setup

  • Adipose-derived stem cells (ASCs) were isolated from the subcutaneous tissue of rabbits and horses.
  • Rabbit ASCs were induced to differentiate into three lineages (tri-lineage differentiation) and then treated with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), fatty acids relevant to cell function and inflammation.
  • Horse ASCs underwent adipogenic, osteogenic, and chondrogenic differentiation, forming multiple experimental groups based on these lineages.
  • Total mRNA was extracted after treatments/differentiation for gene expression analysis.

Selection and Evaluation of Housekeeping Genes

  • The study tested several widely-used housekeeping genes for their stability in both rabbit and horse ASCs under these experimental conditions.
  • Two computational algorithms, RefFinder and NormFinder, were used to analyze and rank the stability of these candidate reference genes.
  • These algorithms assess gene stability by integrating multiple statistical methods, providing a robust evaluation of gene expression consistency.

Key Findings

  • For rabbit ASCs, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed the most stable expression and is recommended as the best endogenous control gene for normalization.
  • For horse ASCs, hypoxanthine phosphoribosyltransferase (HPRT) was found to be the most suitable reference gene due to its consistent expression.
  • Both identified genes remained stable despite different treatments or differentiation protocols, implying their reliability for future experiments.

Implications and Conclusions

  • The research provides a validated baseline for selecting appropriate reference genes in qRT-PCR experiments involving rabbit and horse ASCs.
  • It underscores the necessity of validating reference genes for each specific experimental model rather than assuming universal stability.
  • This individualized approach improves the accuracy and reliability of gene expression studies in stem cell research and potentially other veterinary or biomedical applications involving these species.
  • Researchers working with adipose-derived stem cells from rabbits or horses can use these findings to better design their gene expression analyses, reducing variability and improving reproducibility.

Cite This Article

APA
Ivanova Z, Petrova V, Grigorova N, Vachkova E. (2024). Identification of the Reference Genes for Relative qRT-PCR Assay in Two Experimental Models of Rabbit and Horse Subcutaneous ASCs. Int J Mol Sci, 25(4), 2292. https://doi.org/10.3390/ijms25042292

Publication

International journal of molecular sciences
ISSN: 1422-0067
NlmUniqueID: 101092791
Country: Switzerland
Language: English
Volume: 25
Issue: 4
PII: 2292

Researcher Affiliations

Ivanova, Zhenya
  • Department of Pharmacology, Animal Physiology, Biochemistry and Chemistry, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria.
Petrova, Valeria
  • Department of Pharmacology, Animal Physiology, Biochemistry and Chemistry, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria.
Grigorova, Natalia
  • Department of Pharmacology, Animal Physiology, Biochemistry and Chemistry, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria.
Vachkova, Ekaterina
  • Department of Pharmacology, Animal Physiology, Biochemistry and Chemistry, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria.

MeSH Terms

  • Horses
  • Rabbits
  • Animals
  • Real-Time Polymerase Chain Reaction
  • Genes, Essential
  • Cell Differentiation
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • Adipogenesis
  • Reference Standards
  • Gene Expression Profiling / methods

Grant Funding

  • Project No 09/23, and the Ph.D. Fellowship of Dr. Valeria Petrova / Trakia University

Conflict of Interest Statement

The authors declare no conflicts of interest.

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Citations

This article has been cited 2 times.
  1. Vachkova E, Arnhold S, Petrova V, Heimann M, Koynarski T, Simeonova G, Piperkov P. Transcriptional Factors Related to Cellular Kinetics, Apoptosis, and Tumorigenicity in Equine Adipose-Derived Mesenchymal Stem Cells (ASCs) Are Influenced by the Age of the Donors.. Animals (Basel) 2025 Jun 28;15(13).
    doi: 10.3390/ani15131910pubmed: 40646808google scholar: lookup
  2. Chen YP, Hu CC, Tsai S, Wen ZH, Lin C. Identification of housekeeping gene for future studies exploring effects of cryopreservation on gene expression in shrimp.. Sci Rep 2025 Apr 1;15(1):11046.
    doi: 10.1038/s41598-025-95258-6pubmed: 40169849google scholar: lookup
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