Neither the expression profile nor significance in gene expression of either gene was markedly influenced by normalizing to 18S or the geometric mean of three stable reference genes. In contrast, normalizing Dusp10 and Dusp3 to b-actin resulted in an inaccurate gain or loss of significance, respectively, demonstrating that even small, less than 2-fold variation in reference gene expression can have a significant influence on statistical outcome. Both experiments presented evidence of biological variability as bactin expression approached 2-fold differences between treated and untreated conditions at contiguous time points following stimulation strongly implicating the effect of experimental treatment on relative gene expression. It is also interesting to note that these changes in reference gene expression had a GDC-0199 discernible impact on significance at all time points where b-actin exceeded the DCT # +/20.5 range of suitability. Whether or not the variability of b-actin under these experimental conditions was representative of true biological change in gene expression can be argued, these data clearly demonstrate that careful reference gene validation is critical when considering statistical significance of target genes, especially those that present with marginal changes in gene expression. This investigation further demonstrates that grossly inaccurate conclusions can result from studies where reference genes show marked differences in mRNA abundance that clearly result from treatment or study conditions. Comparing the outcome of normalizing to a variety of conventionally used reference genes that presented with indisputable biological variability, we presented data from two experiments involving three target genes with well-defined roles in adipocyte differentiation where reference gene selection had an unequivocal impact on data interpretation. The first of these experiments examined the expression profile of C/EBPb and cyclin A during the first 24 hrs of differentiation, which is known to include a phase of synchronous cell cycle progression. While the expression profile of either gene was not markedly influenced when normalizing to 18S, the GDC-0449 welldocumented prolonged expression of C/EBPb during this time period as well as the induction of cyclin A at 24 hrs required for S phase progression was completely ablated when normalizing to TfR that presented with its own 6-fold increase in gene expression.