Representational difference analysis of cDNA for the detection of differential gene expression in bacteria: development using a model of iron-regulated gene expression in Neisseria meningitidis

Representational difference analysis of cDNA (cDNA RDA) provides a powerful technique for the identification of specific differences between two mRNA populations, The method has previously been used to analyse differential ge ne expression in eukaryotes, but until now has not been successfully applie d to prokaryotes, A strain of Neisseria meningitidis with a deletion of the iron-regulated lactoferrin-binding protein A (lbpA) gene, grown under iron -replete conditions, and the isogenic parent strain, grown under iron limit ation, were used as a model for developing cDNA RDA for use with bacteria, In this system, the technique should specifically detect the differential e xpression of the lbpA gene in the parent strain, along with other genes who se expression is switched on (or up-regulated) under iron-deficient conditi ons, Since cDNA RDA requires high-quality, representative mRNA, a variety o f methods for the isolation of RNA were evaluated. A triisopropylnaphthalen e sulphonic acid/p-aminosalicylic acid-based technique was found to give th e best results. cDNA was prepared from total RNA isolated from the two N. m eningitidis strains and subjected to an adapted cDNA RDA procedure, The met hod resulted in the amplification of five major PCR products, which include d fragments of the lbpA gene and the iron-regulated RTX-like toxin gene (fr pC), thus validating the technique for use with bacteria.