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 gene expression in eukaryotes, but until now has not been successfully applied to prokaryotes. A strain of Neisseria meningitidis with a deletion of the iron-regulated lactoferrin-binding protein A (IbpA) gene, grown under iron-replete conditions, and the isogenic parent strain, grown under iron limitation, were used as a model for developing cDNA RDA for use with bacteria. In this system, the technique should specifically detect the differential expression of the IbpA gene in the parent strain, along with other genes whose expression is switched on (or up-regulated) under iron-deficient conditions. Since cDNA RDA requires high-quality, representative mRNA, a variety of methods for the isolation of RNA were evaluated. A triisopropylnaphthalene sulphonic acid/ p-aminosalicylic acid-based technique was found to give the best results. cDNA was prepared from total RNA isolated from the two N. meningitidis strains and subjected to an adapted cDNA RDA procedure. The method resulted in the amplification of five major PCR products, which included fragments of the IbpA gene and the iron-regulated RTX-like toxin gene (frpC), thus validating the technique for use with bacteria.
Bowler
Lucas D
LD
School of Biological Sciences, University of Sussex, Brighton BN1 9QG, UK1.
Hubank
Mike
M
Trafford Centre for Medical Research, University of Sussex, Brighton BN1 9RY, UK2.
Spratt
Brian G
BG
Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, Oxford OX1 3PS, UK 3.|School of Biological Sciences, University of Sussex, Brighton BN1 9QG, UK1.
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003603:Cytotoxins; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics*; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D007501:Iron / Q000378:metabolism*; D008565:Membrane Proteins; D009345:Neisseria meningitidis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003603:Cytotoxins; D018076:DNA, Complementary; C112566:LBPA protein, Neisseria meningitidis; D008565:Membrane Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C083530:frpC protein, Neisseria meningitidis; D007501:Iron