Microarrays are a powerful method for the global analysis of gene or protein content and expression, opening up new horizons in molecular and physiological systems. This review focuses on the critical aspects of acquiring meaningful data for analysis following fluorescence-based target hybridisation to arrays. Although microarray technology is adaptable to the analysis of a range of biomolecules (DNA, RNA, protein, carbohydrates and lipids), the scheme presented here is applicable primarily to customised DNA arrays fabricated using long oligomer or cDNA probes. Rather than provide a comprehensive review of microarray technology and analysis techniques, both of which are large and complex areas, the aim of this paper is to provide a restricted overview, highlighting salient features to provide initial guidance in terms of pitfalls in planning and executing array projects. We outline standard operating procedures, which help streamline the analysis of microarray data resulting from a diversity of array formats and biological systems. We hope that this overview will provide practical initial guidance for those embarking on microarray studies.
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T Forster, D Roy, and P Ghazal
Megan N Purpera, Li Shen, Marzieh Taghavi, Heike Münzberg, Roy J Martin, Susan M Hutson, and Christopher D Morrison
Elevation of dietary or brain leucine appears to suppress food intake via a mechanism involving mechanistic target of rapamycin, AMPK, and/or branched chain amino acid (BCAA) metabolism. Mice bearing a deletion of mitochondrial branched chain aminotransferase (BCATm), which is expressed in peripheral tissues (muscle) and brain glia, exhibit marked increases in circulating BCAAs. Here, we test whether this increase alters feeding behavior and brain neuropeptide expression. Circulating and brain levels of BCAAs were increased two- to four-fold in BCATm-deficient mice (KO). KO mice weighed less than controls (25.9 vs 20.4 g, P<0.01), but absolute food intake was relatively unchanged. In contrast to wild-type mice, KO mice preferred a low-BCAA diet to a control diet (P<0.05) but exhibited no change in preference for low- vs high-protein (HP) diets. KO mice also exhibited low leptin levels and increased hypothalamic Npy and Agrp mRNA. Normalization of circulating leptin levels had no effect on either food preference or the increased Npy and Agrp mRNA expression. If BCAAs act as signals of protein status, one would expect reduced food intake, avoidance of dietary protein, and reduction in neuropeptide expression in BCATm-KO mice. Instead, these mice exhibit an increased expression of orexigenic neuropeptides and an avoidance of BCAAs but not HP. These data thus suggest that either BCAAs do not act as physiological signals of protein status or the loss of BCAA metabolism within brain glia impairs the detection of protein balance.
B M McGowan, J S Minnion, K G Murphy, D Roy, S A Stanley, W S Dhillo, J V Gardiner, M A Ghatei, and S R Bloom
Relaxin-3 is a member of the insulin superfamily. It is expressed in the nucleus incertus of the brainstem, which has projections to the hypothalamus. Relaxin-3 binds with high affinity to RXFP1 and RXFP3. RXFP3 is expressed within the hypothalamic paraventricular nucleus (PVN), an area central to the stress response. The physiological function of relaxin-3 is unknown but previous work suggests a role in appetite control, stimulation of the hypothalamic–pituitary–gonadal axis and stress. Central administration of relaxin-3 induces c-fos expression in the PVN and increases plasma ACTH levels in rats. The aim of this study was to investigate the effect of central administration of human relaxin-3 (H3) on the hypothalamic–pituitary–adrenal (HPA) axis in male rodents in vivo and in vitro. Intracerebroventricular (i.c.v) administration of H3 (5 nmol) significantly increased plasma corticosterone at 30 min following injection compared with vehicle. Intra-PVN administration of H3 (1.8–1620 pmol) significantly increased plasma ACTH at 1620 pmol H3 and corticosterone at 180–1620 pmol H3 at 30 min following injection compared with vehicle. The stress hormone prolactin was also significantly raised at 15 min post-injection compared with vehicle. Treatment of hypothalamic explants with H3 (10–1000 nM) stimulated the release of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), but H3 had no effect on the release of ACTH from in vitro pituitary fragments. These results suggest that relaxin-3 may regulate the HPA axis, via hypothalamic CRH and AVP neurons. Relaxin-3 may act as a central signal linking nutritional status, reproductive function and stress.