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

Angela L Riveroll; Sabrina Skyba-Lewin; K Devon Lynn; Glady's Mubyeyi; Ahmad Abd-El-Aziz; Frederick S T Kibenge; Molly J T Kibenge; Alejandro M Cohen; Blanca Esparza-Gonsalez; Laurie McDuffee; William J Montelpare

doi:10.3390/genes14030673

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). 2023 Mar 8;14(3):673. doi: 10.3390/genes14030673.

Affiliations

¹ Department of Applied Human Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A 4P3, Canada.
² Diagnostic Services and Adjunct, Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
³ Department of Biology, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
⁴ Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.
⁵ Biological Mass Spectrometry Core Facility, Dalhousie University, Halifax, NS B3H 4R2, Canada.
⁶ Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada.

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. PPP6R1, CCDC97, and then either ACTB or EPHA2 demonstrated the most stable mRNA levels. Normalizing target gene C_q data with at least three of these RGs simultaneously, as per MIQE guidelines (PPP6R1 and CCDC97 with either ACTB or EPHA2), resulted in congruent conclusions. FABP5 expression was increased in ADs (5.99 and 8.00 fold, p = 0.00002 and p = 0.0003) and in OBs (5.18 and 5.91 fold, p = 0.0011 and p = 0.0023) relative to ADSCs. RUNX2 expression was slightly higher in ADs relative to ADSCs (1.97 and 2.65 fold, p = 0.04 and p = 0.01), but not in OBs (0.9 and 1.03 fold, p = 0.58 and p = 0.91).

Keywords: ACTB; CCDC97; EPHA2; FABP5; PPP6R1; RUNX2; equine adipose-derived mesenchymal stem cell differentiation; gene expression; protein expression; reference genes.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Differentiation / genetics
DNA-Directed RNA Polymerases / metabolism
Gene Expression
Horses / genetics
Mesenchymal Stem Cells* / metabolism
Proteome* / genetics
Proteome* / metabolism
RNA, Messenger / metabolism
RNA-Directed DNA Polymerase / metabolism
Real-Time Polymerase Chain Reaction

Substances

Proteome
RNA-Directed DNA Polymerase
RNA, Messenger
DNA-Directed RNA Polymerases

Grants and funding

This work was funded by the University of Prince Edward INSPIRE Campaign awarded to WM.