Abstract
BackgroundThe 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−.MethodsWe 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.ResultsThe 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.ConclusionsApplying these findings for future gene expression analyses will help elucidate ASC biology.
Highlights
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
Isolation and cultivation of ASCs Human ASCs were isolated from abdominal subcutaneous white adipose tissue samples obtained from four females undergoing routine elective plastic abdominal surgery at the Institute for Plastic and Reconstructive Surgery of the Medical University of Innsbruck
Proliferation and adipogenic differentiation of human ASCs We evaluated candidate reference genes for quantitative RT-PCR-based gene expression studies of proliferating and differentiating human ASCs
Summary
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. 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−. The expression of the adipogenic transcription factor peroxisome proliferator-activated receptor γ2 (PPAR γ2), which is both required and sufficient to drive adipogenesis, is a key event in the induction of the adipogenic differentiation program [9, 10]. To accurately analyze the changes in gene expression during both proliferation and adipogenic differentiation of ASCs, appropriate endogenous controls are needed
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