Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells

Claudia Krautgasser; Markus Mandl; Florian M Hatzmann; Petra Waldegger; Monika Mattesich; Werner Zwerschke

doi:10.1186/s11658-019-0140-6

Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells

Cell Mol Biol Lett. 2019 Feb 15:24:14. doi: 10.1186/s11658-019-0140-6. eCollection 2019.

Authors

Claudia Krautgasser^#¹, Markus Mandl^#¹, Florian M Hatzmann¹, Petra Waldegger¹, Monika Mattesich², Werner Zwerschke¹

Affiliations

¹ 1Division of Cell Metabolism and Differentiation Research, Institute for Biomedical Aging Research, University of Innsbruck, Rennweg 10, A-6020 Innsbruck, Austria.
² 2Department of Plastic and Reconstructive Surgery, Innsbruck Medical University, Anichstraße 35, A-6020 Innsbruck, Austria.

^# Contributed equally.

Abstract

Background: The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1⁺/CD34⁺/CD90⁺/CD105⁺/CD45^-/CD31^-.

Methods: We evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software.

Results: The results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes.

Conclusions: Applying these findings for future gene expression analyses will help elucidate ASC biology.

Keywords: Adipogenesis; Adipose stem cells; Adipose stromal cells; Differentiation; Proliferation; Reference gene.

MeSH terms

Abdominal Fat / cytology*
Abdominal Fat / physiology
Adipogenesis
Cell Proliferation
Gene Expression Profiling / methods*
Gene Expression Profiling / standards
Humans
Real-Time Polymerase Chain Reaction / methods*
Real-Time Polymerase Chain Reaction / standards
Reference Standards
Stromal Cells / physiology