Selection and Validation of Reference Genes for Gene Expression Studies in an Equine Adipose-Derived Mesenchymal Stem Cell Differentiation Model by Proteome Analysis and Reverse-Transcriptase Quantitative Real-Time PCR.
Abstract: Adipose-derived stem cells (ADSCs) are used in tissue regeneration therapies. The objective of this study is to identify stable reference genes (RGs) for use in gene expression studies in a characterized equine adipose-derived mesenchymal stem cell (EADMSC) differentiation model. ADSCs were differentiated into adipocytes (ADs) or osteoblasts (OBs), and the proteomes from these cells were analyzed by liquid chromatography tandem mass spectrometry. Proteins that were stably expressed in all three cells types were identified, and the mRNA expression stabilities for their corresponding genes were validated by RT-qPCR. , , and then either or demonstrated the most stable mRNA levels. Normalizing target gene C data with at least three of these RGs simultaneously, as per MIQE guidelines ( and with either or ), resulted in congruent conclusions. expression was increased in ADs (5.99 and 8.00 fold, = 0.00002 and = 0.0003) and in OBs (5.18 and 5.91 fold, = 0.0011 and = 0.0023) relative to ADSCs. expression was slightly higher in ADs relative to ADSCs (1.97 and 2.65 fold, = 0.04 and = 0.01), but not in OBs (0.9 and 1.03 fold, = 0.58 and = 0.91).
Publication Date: 2023-03-08 PubMed ID: 36980946PubMed Central: PMC10048155DOI: 10.3390/genes14030673Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research is about identifying stable reference genes that can be used within gene expression studies in a specific type of model — an equine adipose-derived mesenchymal stem cell differentiation model. By observing cells that are differentiated into adipocytes or osteoblasts, and then analyzing their proteomes, the study found certain proteins that were stably expressed, and validated the mRNA expression stabilities of their genes.
Objective of the Research
- The main aim of this research was to find stable reference genes for gene expression studies in equine adipose-derived mesenchymal stem cell differentiation models. Reference genes are essential in providing a constant baseline to which gene expression in different samples or under different conditions can be compared.
Methods Used in the Study
- Adipose-derived stem cells were used in the study, differentiated into two different types of cells – adipocytes (fat cells) and osteoblasts (bone cells).
- The proteomes (the entire set of proteins expressed by a genome, cell, tissue, or organism at a certain time) of these cells were studied using liquid chromatography tandem mass spectrometry, a highly sensitive technique used to identify and characterize proteins.
- The researchers identified proteins that were stably expressed in all three cell types and then validated the mRNA expression stabilities of these proteins’ corresponding genes using reverse-transcriptase quantitative real-time PCR (RT-qPCR) – a method commonly used to measure gene expression.
Key Findings of the Study
- The study discovered certain proteins that were consistently and stably expressed across the three types of cells. The mRNA levels for these corresponding genes demonstrated high stability.
- When normalizing target gene data with at least three of these reference genes simultaneously, the study obtained congruent conclusions.
- The research also identified that gene C expression was significantly increased in adipocytes and osteoblasts relative to adipose-derived stem cells. A slightly higher gene C expression was also observed in adipocytes compared to adipose-derived stem cells, but there was no significant difference in osteoblasts.
Implications of the Research
- The findings of this study are significant as they aid in providing better research tools for gene expression studies in equine adipose-derived mesenchymal stem cells. By identifying stable reference genes, researchers can have more accurate comparisons and analysis in future research.
- Furthermore, the particular expression patterns of gene C observed in this study could provide interesting insights into cellular differentiation and might be useful for therapeutic applications in tissue regeneration.
Cite This Article
APA
Riveroll AL, Skyba-Lewin S, Lynn KD, Mubyeyi G, Abd-El-Aziz A, Kibenge FST, Kibenge MJT, Cohen AM, Esparza-Gonsalez B, McD￮ L, Montelpare WJ.
(2023).
Selection and Validation of Reference Genes for Gene Expression Studies in an Equine Adipose-Derived Mesenchymal Stem Cell Differentiation Model by Proteome Analysis and Reverse-Transcriptase Quantitative Real-Time PCR.
Genes (Basel), 14(3), 673.
https://doi.org/10.3390/genes14030673 Publication
Researcher Affiliations
- Department of Applied Human Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A 4P3, Canada.
- Diagnostic Services and Adjunct, Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
- Department of Biology, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
- Department of Biology, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
- Department of Biology, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
- Department of Biology, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
- Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
- Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
- Biological Mass Spectrometry Core Facility, Dalhousie University, Halifax, NS B3H 4R2, Canada.
- Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
- Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
- Department of Applied Human Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A 4P3, Canada.
MeSH Terms
- Animals
- Horses / genetics
- Proteome / genetics
- Proteome / metabolism
- RNA-Directed DNA Polymerase / metabolism
- Real-Time Polymerase Chain Reaction
- Cell Differentiation / genetics
- Mesenchymal Stem Cells / metabolism
- Gene Expression
- RNA, Messenger / metabolism
- DNA-Directed RNA Polymerases / metabolism
Conflict of Interest Statement
The authors declare no conflict of interest.
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