Generation of microarrays for the study of gene expression patterns in Ralstonia solanacearum

This work represents the first steps in the development of functional genomic methods to identify variably expressed genes involved in the regulation of host specificity within Ralstonia solanacearum. Microarrays were designed that contained a subset of the genetic complement of R. solanacearum. These were spotted out onto glass slides using a commercial microarrayer, with sufficient replicates of each fragment included to allow for statistically significant expression level differences to be measured. Extraction of bacterial mRNA from in vitro cultures and infected plant material was performed using commercial total RNA extraction kits. Problems encountered included obtaining sufficient amounts of bacterial RNA in total plant extract, evaluating mRNA extraction efficiency, and reverse transcription of bacterial mRNA. This work has given us the techniques and experience in the use of microarrays to conduct functional genomic studies into the differential expression of a large number of functionally uncharacterised genes from the recently published genomic sequence of R. solanacearum.