title: In vivo labeling and specific magnetic bead separation of RNA for biofilm characterization and stress-induced gene expression analysis in bacteria.
abstract: The method of in vivo labeling and separation of bacterial RNA was developed as an approach to elucidating the stress response of natural bacterial populations. This technique is based on the incorporation of digoxigenin-11-uridine-5'-triphosphate (DIG-11-UTP) in the RNA of active bacteria. The digoxigenin fulfills a dual role as a label of de novo synthesized RNA and a target for magnetic bead separation from a total RNA extract. Depending on the growth conditions and the population's composition, the assembly rate of DIG-11-UTP ranged from 1.2% to 12.5% of the total RNA in gram-positive and gram-negative reference bacteria as well as in natural biofilms from drinking water, surface water, and lake sediment. Separation of DIG-RNA from total RNA extracts was performed with a biotinylated anti-digoxigenin antibody and streptavidin-functionalized magnetic particles. The average separation yield from total RNA extracts was about 95% of labeled RNA. The unspecific bindings of non-labeled nucleic acids were smaller than 0.2%, as was evaluated by spiking experiments with an unmarked DNA amplicon. Applicability of the method developed was demonstrated by rRNA-directed PCR-DGGE population analysis of natural biofilms and expression profiling of two stress-induced genes (vanA and rpoS) in reference bacteria.
authors.affiliation: Karlsruhe Institute of Technology - KIT (former: Forschungszentrum Karlsruhe), Institute of Functional Interfaces (IFG), Microbiology of Natural and Technical Interfaces Department, P.O. Box 3640, D-76021 Karlsruhe, Germany.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Stankiewicz
authors.forename: Andrea
authors.initials: A
authors.lastname: Gold
authors.forename: Yousra
authors.initials: Y
authors.lastname: Yüksel
authors.forename: Sonja
authors.initials: S
authors.lastname: Berensmeier
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwartz
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000378:metabolism; D018441:Biofilms; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D004076:Digoxigenin / Q000031:analogs & derivatives / Q000737:chemistry; D016984:Enterococcus faecium / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D008280:Magnetics; D008863:Microspheres; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D003856:Deoxyuracil Nucleotides; D006360:Heat-Shock Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; C070202:digoxigenin-11-deoxyuridine triphosphate; D004076:Digoxigenin