A universal reference sample derived from clone vector for improved detection of differential gene expression.

BACKGROUND: Using microarrays by co-hybridizing two samples labeled with different dyes enables differential gene expression measurements and comparisons across slides while controlling for within-slide variability. Typically one dye produces weaker signal intensities than the other often causing signals to be undetectable. In addition, undetectable spots represent a large problem for two-color microarray designs and most arrays contain at least 40% undetectable spots even when labeled with reference samples such as Stratagene's Universal Reference RNAs. RESULTS: We introduce a novel universal reference sample that produces strong signal for all spots on the array, increasing the average fraction of detectable spots to 97%. Maximizing detectable spots on the reference image channel also decreases the variability of microarray data allowing for reliable detection of smaller differential gene expression changes. The reference sample is derived from sequence contained in the parental EST clone vector pT7T3D-Pac and is called vector RNA (vRNA). We show that vRNA can also be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This reference sample can be made inexpensively in large quantities as a renewable resource that is consistent across experiments. CONCLUSION: Results of this study show that vRNA provides a useful universal reference that yields high signal for almost all spots on a microarray, reduces variation and allows for comparisons between experiments and laboratories. Further, it can be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This type of reference allows for detection of small changes in differential expression while reference designs in general allow for large-scale multivariate experimental designs. vRNA in combination with reference designs enable systems biology microarray experiments of small physiologically relevant changes.

Chronic alcohol exposure alters transcription broadly in a key integrative brain nucleus for homeostasis: the nucleus tractus solitarius.

Chronic exposure to alcohol modifies physiological processes in the brain, and the severe symptoms resulting from sudden removal of alcohol from the diet indicate that these modifications are functionally important. We investigated the gene expression patterns in response to chronic alcohol exposure (21-28 wk) in the rat nucleus tractus solitarius (NTS), a brain nucleus with a key integrative role in homeostasis and cardiorespiratory function. Using methods and an experimental design optimized for detecting transcriptional changes less than twofold, we found 575 differentially expressed genes. We tested these genes for significant associations with physiological functions and signaling pathways using Gene Ontology terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, respectively. Chronic alcohol exposure resulted in significant NTS gene regulation related to the general processes of synaptic transmission, intracellular signaling, and cation transport as well as specific neuronal functions including plasticity and seizure behavior that could be related to alcohol withdrawal symptoms. The differentially expressed genes were also significantly enriched for enzymes of lipid metabolism, glucose metabolism, oxidative phosphorylation, MAP kinase signaling, and calcium signaling pathways from KEGG. Intriguingly, many of the genes we found to be differentially expressed in the NTS are known to be involved in alcohol-induced oxidative stress and/or cell death. The study provides evidence of very extensive alterations of physiological gene expression in the NTS in the adapted state to chronic alcohol exposure.

Dynamic transcriptomic response to acute hypertension in the nucleus tractus solitarius.

Baroreceptor afferents project to the cardiovascular region of the nucleus tractus solitarius (cvNTS), and their cvNTS target neurons may play a role in governing the sensitivity and operating range of the arterial baroreceptor reflex (baroreflexes). Recent studies have shown differential gene and protein expression in the cvNTS in response to changed arterial pressure. However, the extent of these responses is unknown. Therefore, we collected differential global gene expression data in a time series following acute hypertension in awake, freely moving rats. To acquire statistically significant results and place them in functional context, we overcame several quality control requirements and developed novel analytical approaches. The physiologically new findings from the study are that acute hypertension causes very extensive, time-varying gene regulatory changes, many involving neuronal function-specific genes and systems of genes. We use standard genomic analysis methods to manage the large data sets and to develop results such as heat maps to examine patterns and clusters in the gene regulation. We used the Gene Ontology categories to provide functional context. To place our findings in the context of the relevant literature, we developed two graphical representations of the networks implicated, linking receptors and channels to signaling pathways. The results point to the multivariate complexity of the response and implicate a group of receptors as candidates for mediating nucleus tractus solitarius baroreflex function in hypertension by identifying concurrent upregulation of receptor genes. We were able to make transcription factor binding predictions and record dysregulation of heart rate correlated with the transcriptional response.