title: Affinity capillary electrophoresis with magnetic beads for multiplex quantitative analysis of bacterial 16S rRNA.
abstract: We have developed a novel method for microbial community analysis of bacterial 16S rRNAs based on affinity capillary electrophoresis using 16S rRNA-conjugated magnetic beads. We called this method magnetic beads affinity capillary electrophoresis (MB-ACE) which can be used for sequential and quantitative analysis of 16S rRNA. In this method, RNA extracted from a microbial community is biotin-modified and mixed with streptavidin-modified paramagnetic beads. This mixture is then injected into a capillary and localized in the middle of the capillary using a magnet held adjacent to the capillary. Subsequently, a fluorescent-labeled probe to detect the target 16S rRNA is injected into the capillary, and voltage is applied. The probe trapped on the RNA is dissociated by formamide and detected at its anodic end by measuring the fluorescence. Next, another fluorescent probe is injected, and thus the target 16S rRNA in the sample is quantified one by one. MB-ACE was used for the quantification of the 16S rRNAs of Escherichia coli and Pseudomonas putida in samples that were prepared by mixing RNA extracted from activated sludge and 16S rRNAs prepared by in vitro transcription. The two types of 16S rRNAs were quantified, indicating that MB-ACE can be used for sequential quantitative analysis of bacterial 16S rRNAs.
authors.affiliation: Department of Chemical Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Adachi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Yamaguchi
authors.forename: Makoto
authors.initials: M
authors.lastname: Nakashige
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kanagawa
authors.forename: Masaki
authors.initials: M
authors.lastname: Torimura
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tsuneda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sekiguchi
authors.forename: Naohiro
authors.initials: N
authors.lastname: Noda
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D016958:Pseudomonas putida / Q000378:metabolism; D012329:RNA, Bacterial / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D012722:Sewage / Q000032:analysis
chemical_list: D005456:Fluorescent Dyes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012722:Sewage