Validation of endogenous controls for gene expression studies in human adipocytes and preadipocytes

Quantitative gene expression protocols require adequate controls to monitor intersample variation. Quantitative approaches to describe relative change s 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 h ousekeeping genes are widely used as endogenous controls. However, literatu re reports suggest changes in gene expression of typical housekeeping genes (e.g. GAPDH, beta -actin, 18S rRNA) upon hormonal stimulation or during ad ipogenic differentiation. Thus, we tested the influence of 6 hormones and a dipogenic differentiation on gene expression levels of 11 commonly used hou sekeeping genes in primary cultured mature human ;adipocytes and preadipocy tes. Using the TaqMan((R)) RT-PCR technique and "Human Endogenous Control A ssays" (PE Biosystems), we found several housekeeping genes with at least t wice the difference in expression levels between stimulated and unstimulate d cells (such as acidic ribosomal protein, beta -actin, beta (2)-Microglobu lin and beta -glucuronidase). Only GAPDH and transferrin receptor gene expr ession levels did not change under any of the stimuli tested, thus appeared best suited for gene expression studies in human adipose cells across a wi de range of experimental settings.