BackgroundRT-qPCR is a sensitive and increasingly used method for gene expression quantification. To normalize RT-qPCR measurements between samples, most laboratories use endogenous reference genes as internal controls. There is increasing evidence, however, that the expression of commonly used reference genes can vary significantly in certain contexts.ResultsUsing the Genevestigator database of normalized and well-annotated microarray experiments, we describe the expression stability characteristics of the transciptomes of several organisms. The results show that a) no genes are universally stable, b) most commonly used reference genes yield very high transcript abundances as compared to the entire transcriptome, and c) for each biological context a subset of stable genes exists that has smaller variance than commonly used reference genes or genes that were selected for their stability across all conditions.ConclusionWe therefore propose the normalization of RT-qPCR data using reference genes that are specifically chosen for the conditions under study. RefGenes is a community tool developed for that purpose. Validation RT-qPCR experiments across several organisms showed that the candidates proposed by RefGenes generally outperformed commonly used reference genes. RefGenes is available within Genevestigator at http://www.genevestigator.com.

27 kDa accessory HIV and simian immunodeficiency virus (SIV) protein necessary for efficient virus replication and high viral load, has for several years been considered as a progression factor to AIDS (Greene and Peterlin, 2002). According to several excellent reviews, a variety of diverse functions including downregulation of cell-surface molecules have been assigned to the protein (Das and Jameel, 2005;Piguet and Trono, 1999;Roeth and Collins, 2006). Moreover, HIV-associated dementia (HAD) correlates with infiltration of monocytes into the brain. The accessory HIV-1 negative factor (Nef) protein, which modulates several signaling pathways, is constitutively present in persistently infected astroctyes. We demonstrated that monocytes responded with chemotaxis when subjected to cell culture supernatants of nef-expressing astrocytic U251MG cells. Using a protein array, we identified CC chemokine ligand 2/monocyte chemotactic protein-1 (CCL2/MCP-1) as a potential chemotactic factor mediating this phenomenon. CCL2/MCP-1 upregulation by Nef was further confirmed by ribonuclease protection assay, RT-PCR and ELISA. By applying neutralizing antibodies against CCL2/MCP-1 and using CCR2-deficient monocytes, we confirmed CCL2/MCP-1 as the exclusive factor secreted by nefexpressing astrocytes capable of attracting monocytes. Additionally, we showed that Nef-induced CCL2/MCP-1 expression depends on the myristoylation moiety of Nef and requires functional calmodulin. In summary, we suggest that Nef-induced CCL2/MCP-1 expression in astrocytes contributes to infiltration of monocytes into the brain, and thereby to progression of HAD.

Prebiotics are suggested as an alternative to antibiotics in animal rearing. Fermentable substances such as inulin or lactulose have been proposed to stimulate the immune system and health by modulation of the intestinal flora and its fermentation products. In this study, effects of inulin and lactulose on the intestinal health and hematology of calves have been investigated. Both prebiotics significantly decreased thrombocyte counts in peripheral blood. Only inulin was able to increase hemoglobin concentration and hematocrit. Total leukocyte count was decreased by lactulose while both prebiotics tended to lower monocyte proportions. mRNA expression of inflammation-related markers in the intestine was also affected by both prebiotics hinting at a decreased inflammatory status. This may be due to a possible decrease in intestinal pathogen load that remains to be verified. Only mRNA amounts of interleukin 8 were increased by lactulose in mesenteric lymph nodes. In the ileum, expression of a proliferation marker was increased by inulin while an apoptosis-related gene was increased by both prebiotics. The results of this study show a clear effect of prebiotics on certain parameters associated with animal health and performance that remain to be studied in detail in future investigations.