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International journal of molecular sciences
Feb 14, 2024;25 (4):

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

Zhenya Ivanova, Valeria Petrova, Natalia Grigorova, Ekaterina Vachkova
Author Information
  1. Zhenya Ivanova: Department of Pharmacology, Animal Physiology, Biochemistry and Chemistry, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria.
  2. Valeria Petrova: Department of Pharmacology, Animal Physiology, Biochemistry and Chemistry, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria. ORCID
  3. Natalia Grigorova: Department of Pharmacology, Animal Physiology, Biochemistry and Chemistry, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria. ORCID
  4. Ekaterina Vachkova: Department of Pharmacology, Animal Physiology, Biochemistry and Chemistry, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria.
PMID: 38396967 DOI: 10.3390/ijms25042292

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.

Keywords

adipose tissue endogenous control equine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) hypoxanthine-guanine phosphoribosyltransferase (HPRT) rabbit

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Grants

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

MeSH Term

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

Chemicals

Glyceraldehyde-3-Phosphate Dehydrogenases
Journal Article

Word Cloud

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