Despite the publication of the MIQE (Minimum Information for Publication of Quantitative. Real-Time PCR Experiments) guidelines , incorrect presumptions that “traditional” reference genes are stably expressed, without reported validation under specific experimental conditions, are widespread among gene expression studies . The stability of traditional references genes, such as Gapdh and Actinb, has previously been called into question [39–41]. In agreement, the current study further highlights the pitfalls of using such an approach for the selection of reference genes for data normalisation. The commonly used reference gene, Actinb, although stably expressed at 24 h post-LPS stimulation (ranked third and fourth, by NormFinder and BestKeeper, respectively), was found to be one of the most highly regulated genes when cells were exposed to LPS for 6 h (ranked in the lowest four by all three programs). Similarly, levels of Hprt gene expression at 6 h post LPS-treatment were stable, however, at 24 h, expression levels were down-regulated by exposure to LPS. These results demonstrate that reference gene expression levels are modulated by their cellular conditions, and highlight the requirement that all genes must be tested for expression stability under the specific experimental conditions being studied.