Affinity capillary electrophoresis with magnetic beads for multiplex quantitative analysis of bacterial 16S rRNA

Ken Adachi, Masahiro Yamaguchi, Makoto Nakashige, Takahiro Kanagawa, Masaki Torimura, Satoshi Tsuneda, Yuji Sekiguchi, Naohiro Noda

    Research output: Contribution to journalArticle

    6 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)662-667
    Number of pages6
    JournalJournal of Bioscience and Bioengineering
    Volume107
    Issue number6
    DOIs
    Publication statusPublished - 2009 Jun

    Fingerprint

    Capillary electrophoresis
    Capillary Electrophoresis
    RNA
    Chemical analysis
    Fluorescent Dyes
    Pseudomonas putida
    Streptavidin
    Magnets
    Transcription
    Biotin
    Sewage
    Escherichia coli
    Fluorescence
    Electric potential

    Keywords

    • 16S rRNA
    • Affinity capillary electrophoresis
    • Magnetic beads
    • Microbial community analysis
    • Quantitative detection

    ASJC Scopus subject areas

    • Biotechnology
    • Applied Microbiology and Biotechnology
    • Bioengineering

    Cite this

    Affinity capillary electrophoresis with magnetic beads for multiplex quantitative analysis of bacterial 16S rRNA. / Adachi, Ken; Yamaguchi, Masahiro; Nakashige, Makoto; Kanagawa, Takahiro; Torimura, Masaki; Tsuneda, Satoshi; Sekiguchi, Yuji; Noda, Naohiro.

    In: Journal of Bioscience and Bioengineering, Vol. 107, No. 6, 06.2009, p. 662-667.

    Research output: Contribution to journalArticle

    Adachi, Ken ; Yamaguchi, Masahiro ; Nakashige, Makoto ; Kanagawa, Takahiro ; Torimura, Masaki ; Tsuneda, Satoshi ; Sekiguchi, Yuji ; Noda, Naohiro. / Affinity capillary electrophoresis with magnetic beads for multiplex quantitative analysis of bacterial 16S rRNA. In: Journal of Bioscience and Bioengineering. 2009 ; Vol. 107, No. 6. pp. 662-667.
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