NR AXVL
AU Roupaka,S.; Gossner,A.; Murphy,L.; Sales,J.; Hunter,N.; Hopkins,J.
TI Functional Genomic Analysis of Preclinical Scrapie in Sheep Using EST Microarrays
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Pathology and Pathogenesis P03.183
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB Functional genomics of peripheral tissues of scrapie infected sheep has been used for the identification of surrogate markers of preclinical infection as well as understanding disease pathogenesis. Our study used a 20,000 EST microarray to assess differential gene expression in spleen follicular dendritic cells of VRQ/VRQ New Zealand Cheviot sheep taken at 75 days post infection with SSBP/1 scrapie. Normalization was carried out on each chip separately using the print-tip loess method from the Limma package of the Bioconductor project. Analysis of these microarray data indicated downregulation of the most significant differentially expressed genes in the diseased samples for the majority of the genes the analysed. A series of candidate genes were selected from the genes differentially expressed in control (mock-infected) and infected animals. PCR primers were designed, products were cloned and sequenced to verify gene identity and a real time quantitative RT-PCR assay was optimised in order to validate the microarray data. A panel of housekeeping genes were assayed for expression stability and those exhibiting the least variation were selected as normalization controls. In order to assess the utility of each candidate as a surrogate marker of infection the expression level of each gene was quantified in spleen samples taken from SSBP/1-infected and mock-infected VRQ/VRQ sheep at 6 time points, from pre-clinical through to clinical disease (10, 25, 50, 75, 100, 125 d.p.i). Fully validated candidates have now been used to assess their use as diagnostic markers by screening blood buffy coat samples from the same animals.
AD S. Roupaka, L. Murphy, J. Hopkins, Royal (Dick) School of Veterinary Studies, UK; A. Gossner, Hokkaido University, Japan; J. Sales, Biomathematics & Statistics (Scotland), UK; N. Hunter, Roslin Institute, Neuropathogenesis Unit, UK
SP englisch
PO Schottland