Project description:Combining or pooling individual samples when carrying out transcript profiling using microarrays is a fairly common means to reduce both the cost and complexity of data analysis. However, pooling does not allow for statistical comparison of changes between samples and can result in a loss of information. Because a rigorous comparison of the identified expression changes from the two approaches has not been reported, we compared the results for hepatic transcript profiles from pooled vs. individual samples. Hepatic transcript profiles from a single-dose time-course rat study in response to the prototypical toxicants Clofibrate [CAS:637-07-0;CHEBI:3750], Diethylhexyl phthalate DEHP [CAS:117-81-7;CHEBI:17243], and valproic acid VPA [CAS:1069-66-5;CHEBI:9925],were evaluated. Approximately 50% more transcript expression changes were observed in the individual (statistical) analysis compared with the pooled analysis. While the majority of these changes were less than twofold in magnitude (~80%), a substantial number were greater than twofold (~20%). Transcript changes unique to the individual analysis were confirmed by quantitative RT-PCR, while all the changes unique to the pooled analysis did not confirm. The individual analysis identified more hits per biological pathway than the pooled approach. Many of the transcripts identified by the individual analysis were novel findings and may contribute to a better understanding of molecular mechanisms of these compounds. Furthermore, having individual animal data provided the opportunity to correlate changes in transcript expression to phenotypes (i.e., histology) observed in toxicology studies. The two approaches were similar when clustering methods were used despite the large difference in the absolute number of transcripts changed. In summary, pooling reduced resource requirements substantially, but the individual approach enabled statistical analysis that identified more gene expression changes to evaluate mechanisms of toxicity. An individual animal approach becomes more valuable when the overall expression response is subtle and/or when associating expression data to variable phenotypic responses.
Project description:Array data from a primary rat hepatocyte model toxicology system dosed with carbon tetrachloride has been produced on Codelink microarray platforms using three starting qualities of RNA ("high" (RIN: 9.0), "medium" (RIN: 5.0) and "low" (RIN: 2.5) quality, assessed by RIN number).
Project description:Viral infections and local production of IFNgamma might contribute to beta-cell dysfunction/death in Type 1 diabetes. Double stranded RNA accumulates in the cytosol of viral-infected cells, and exposure of purified rat beta cells to dsRNA (polyinosinic-polycytidylic acid, PIC) in combination with IFNgamma results in beta-cell dysfunction and apoptosis. To elucidate the molecular mechanisms involved in PIC + IFNgamma-effects, we determined the global profile of genes modified by these agents in primary rat beta cells. FACS-purified rat beta cells were cultured for 6 or 24 h in control condition or with IFNgamma, PIC or a combination of both agents. The gene expression profile was analyzed in duplicate with the Affymetrix RG U34A microarray.
Project description:We investigated genome-wide changes in mRNA translation in Arabidopsis thaliana suspension cell cultures exposed to brief perids of two types of stress: elevated temperature (37 degree_C) and high salinity (200 mM NaCl). To this end, we subjected polysomal RNA and non-polysomal RNA from sucrose gradient fractionated cell lysates to the co-hybridization on Agilent Arabidopsis 3 Oligo Microarrays. The ratio of signal intensities (polysomal RNA: non-polysomal RNA) was used as an indicator of the translation state for each transcript. To inspect coordination of changes in translational profiles with transcriptional profiles, we also isolated total RNAs from the same cells used for translational profiling experiments and investigated changes in accumulated transcript levels in response to each stress using the microarray. Two biological replicates were analyzed.