Horm Metab Res 2001; 33(10): 625-627
DOI: 10.1055/s-2001-17911
Innovative Methods
© Georg Thieme Verlag Stuttgart · New York

Validation of Endogenous Controls for Gene Expression Studies in Human Adipocytes and Preadipocytes

K. Gorzelniak, J. Janke, S. Engeli, A. M. Sharma
  • Franz-Volhard-Klinik, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Germany
  • and Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany
Further Information

Publication History

Publication Date:
18 October 2001 (online)

Quantitative gene expression protocols require adequate controls to monitor intersample variation. Quantitative approaches to describe relative changes in gene expression use endogenous controls - ”housekeeping” genes. Given the low amounts of mRNA in fat cells, RT-PCR is the method of choice, and housekeeping genes are widely used as endogenous controls. However, literature reports suggest changes in gene expression of typical housekeeping genes (e. g. GAPDH, β-actin, 18S rRNA) upon hormonal stimulation or during adipogenic differentiation. Thus, we tested the influence of 6 hormones and adipogenic differentiation on gene expression levels of 11 commonly used housekeeping genes in primary cultured mature human adipocytes and preadipocytes. Using the TaqMan® RT-PCR technique and “Human Endogenous Control Assays” (PE Biosystems), we found several housekeeping genes with at least twice the difference in expression levels between stimulated and unstimulated cells (such as acidic ribosomal protein, β-actin, β2 -microglobulin and β-glucuronidase). Only GAPDH and transferrin receptor gene expression levels did not change under any of the stimuli tested, thus appeared best suited for gene expression studies in human adipose cells across a wide range of experimental settings.

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Prof. Dr. med. Arya M. Sharma

Department of Nephrology and Hypertension
Franz-Volhard-Klinik
Universitätsklinikum Charité
Humbold Universität zu Berlin

Wiltbergstrasse 50
13125 Berlin-Buch
Germany


Phone: + 49 (30) 9417 2203

Fax: + 49 (30) 9417 2206

Email: sharma@fvk-berlin.de

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