Approaches to Differential Gene Expression Analysis in Atherosclerosis

Today’s rapid development of powerful tools for gene expression analysis provides unprecedented resources for elucidating complex molecular events. The objective of this work has been to apply, combine and evaluate tools for analysis of differential gene expression using atherosclerosis as a model system. First, an optimised solid-phase protocol for representational difference analysis (RDA) was applied to two in vitro model systems. Initially, The RDA enrichment procedure was investigated by shotgun cloning and sequencing of successive difference products. In the subsequent steps, combinations of RDA and microarray analysis were used to combine the selectivity and sensitivity of RDA with the high-throughput nature of microarrays. This was achieved by immobilization of RDA clones onto microarrays dedicated for gene expression analysis in atherosclerosis as well as hybridisation of labelled RDA products onto global microarrays containing more than 32,000 human clones. Finally, RDA was applied for the investigation of the focal localisation of atherosclerotic plaques in mice using in vivo tissue samples as starting material. A large number of differentially expressed clones were isolated and confirmed by real time PCR. A very diverse range of gene fragments was identified in the RDA products especially when they were screened with global microarrays. However, the microarray data also seem to contain some noise which is a general problem using microarrays and should be compensated for by careful verification of the results. Quite a large number of candidate genes related to the atherosclerotic process were found by these studies. In particular several nuclear receptors with altered expression in response to oxidized LDL were identified and deserve further investigation. Extended functional annotation does not lie within the scope of this thesis but raw data in the form of novel sequences and accession numbers of known sequences have been made publicly available in GenBank. Parts of the data are also available for interactive exploration on-line through an interactive software tool. The data generated thus constitute a base for new hypotheses to be tested in the field of atherosclerosis.