|Jahn, Courtney - UW MADISON|
|Charkowski, Amy - UW MADISON|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 9, 2008
Publication Date: July 15, 2008
Citation: Jahn, C.E., Charkowski, A.O., Willis, D.K. 2008. Evaluation of isolation methods for bacterial RNA quantitation in Dickeya dadantii. Applied and Environmental Microbiology. 75(2):318-324. Interpretive Summary: We are studying the mechanism by which bacteria are able to cause plant disease. The enteric phytopathogen Dickeya dadantii causes wilting and soft rot in a wide range of plants, including many weed species, ornamental plants, and economically important crops. D. dadantii produces a variety of cell wall-degrading enzymes, such as pectate lyases and cellulases, that cause tissue maceration and these enzymes are the primary pathogenicity factor of this enterobacterium. We hope to gain an understanding of the molecular biology of the genes and gene products that are required for the disease process by the bacteria. Ultimately, this understanding will provide information of use to breeders and plant molecular biologists that will enable the development of plant lines resistant to disease organisms. This manuscript describes a technical advance in the isolation and quality assessment of RNA from this plant pathogen that is suitable for downstream application such as quantification of virulence gene expression.
Technical Abstract: Dickeya dadantii is a difficult source for RNA of a sufficient quality for real-time qRT-PCR analysis of gene expression. Three RNA isolation methods were evaluated for their ability to produce high-quality RNA from this bacterium. Bacterial lysis with Trizol using standard protocols consistently gave low RNA yields with this organism. Higher yields due to improved bacterial cells lysis was achieved with an added hot SDS incubation step, but RNA quality was low as determined by the RNA Integrity Number (RIN). Contaminating DNA remained a problem with the hot SDS-Trizol method; RNA samples required repeated, rigorous DNase treatments to reduce DNA contamination to levels sufficient for successful real-time qRT-PCR. A hot SDS-hot phenol RNA method gave the highest RNA quality and required only two DNase treatments to remove DNA. The assessment of RNA integrity using the Agilent 2100 BioAnalyzer was critical for obtaining meaningful gene expression data. RIN values below 7.0 resulted in high variation and loss of statistical significance when gene expression was analyzed by real-time qRT-PCR. This work provides guidelines for RNA isolation and quality assessment that will valuable for gene expression studies in a wide range of bacteria.