Selection and Validation of Reference Genes for Gene Expression Studies in an Equine Adipose-Derived Mesenchymal Stem...

Area of Science:

• Biotechnology
• Stem Cell Biology
• Molecular Biology

Background:

• Adipose-derived stem cells (ADSCs) are crucial for tissue regeneration.
• Accurate gene expression analysis in equine ADSCs (EADSCs) requires validated reference genes.
• Understanding EADSC differentiation into adipocytes and osteoblasts is key for therapeutic applications.

Purpose of the Study:

• To identify stable reference genes for quantitative RT-qPCR in equine ADSC differentiation models.
• To ensure reliable gene expression data for EADSC research and therapeutic development.

Main Methods:

• ADSCs were differentiated into adipocytes (ADs) and osteoblasts (OBs).
• Proteomic analysis using liquid chromatography tandem mass spectrometry identified stably expressed proteins.
• mRNA expression stability of corresponding genes was validated using RT-qPCR.

Main Results:

• PPP6R1, CCDC97, ACTB, and EPHA2 were identified as the most stable reference genes.
• Normalizing target gene data with combinations of these reference genes (e.g., PPP6R1 and CCDC97 with ACTB or EPHA2) yielded congruent results, adhering to MIQE guidelines.
• FABP5 expression significantly increased in both ADs and OBs compared to ADSCs.
• RUNX2 expression was slightly elevated in ADs but not in OBs relative to ADSCs.

Conclusions:

• PPP6R1, CCDC97, ACTB, and EPHA2 are validated as reliable reference genes for EADSC differentiation studies.
• Using these reference genes ensures accurate normalization and interpretation of gene expression data in equine stem cell research.
• The findings support the MIQE guidelines for robust gene expression analysis in regenerative medicine.