title: Development of a single-step subtraction method for eukaryotic 18S and 28S ribonucleic acids.
abstract: The abundance of mammalian 18S and 28S ribosomal RNA can decrease the detection sensitivity of bacterial or viral targets in complex host-pathogen mixtures. A method to capture human RNA in a single step was developed and characterized to address this issue. For this purpose, capture probes were covalently attached to magnetic microbeads using a dendrimer linker and the solid phase was tested using rat thymus RNA (mammalian components) with Escherichia coli RNA (bacterial target) as a model system. Our results indicated that random capture probes demonstrated better performance than specific ones presumably by increasing the number of possible binding sites, and the use of a tetrame-thylammonium-chloride (TMA-Cl-) based buffer for the hybridization showed a beneficial effect in the selectivity. The subtraction efficiency determined through real-time RT-PCR revealed capture-efficiencies comparable with commercially available enrichment kits. The performance of the solid phase can be further fine tuned by modifying the annealing time and temperature.
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington DC 20375, USA. marie.j.archer@gmail.com
authors.forename: Marie J
authors.initials: MJ
authors.lastname: Archer
authors.forename: Baochuan
authors.initials: B
authors.lastname: Lin
mesh_terms: D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D056890:Eukaryota / Q000378:metabolism*; D008280:Magnetics / Q000379:methods*; D008863:Microspheres; D009693:Nucleic Acid Hybridization / Q000379:methods; D012313:RNA / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000302:isolation & purification*; D012339:RNA, Ribosomal, 28S / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats; D013950:Thymus Gland / Q000378:metabolism
chemical_list: D012329:RNA, Bacterial; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D012313:RNA