File(s) not publicly available
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
journal contribution
posted on 2023-06-08, 08:21 authored by Lucas Bowler, Mike Hubank, Brian SprattRepresentational 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 (lbpA) 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 lbpA 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 lbpA gene and the iron-regulated RTX-like toxin gene (frpC), thus validating the technique for use with bacteria.
History
Publication status
- Published
Journal
MicrobiologyISSN
1350-0872Publisher
Society for General MicrobiologyVolume
145Page range
3529-3537ISBN
1350-0872Department affiliated with
- Biochemistry Publications
Full text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2012-02-06Usage metrics
Categories
No categories selectedKeywords
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC