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 language | English |
|---|---|
| Pages (from-to) | 662-667 |
| Number of pages | 6 |
| Journal | Journal of Bioscience and Bioengineering |
| Volume | 107 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 2009 Jun |
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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 journal › Article
}
TY - JOUR
T1 - Affinity capillary electrophoresis with magnetic beads for multiplex quantitative analysis of bacterial 16S rRNA
AU - Adachi, Ken
AU - Yamaguchi, Masahiro
AU - Nakashige, Makoto
AU - Kanagawa, Takahiro
AU - Torimura, Masaki
AU - Tsuneda, Satoshi
AU - Sekiguchi, Yuji
AU - Noda, Naohiro
PY - 2009/6
Y1 - 2009/6
N2 - 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.
AB - 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.
KW - 16S rRNA
KW - Affinity capillary electrophoresis
KW - Magnetic beads
KW - Microbial community analysis
KW - Quantitative detection
UR - http://www.scopus.com/inward/record.url?scp=65549084520&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=65549084520&partnerID=8YFLogxK
U2 - 10.1016/j.jbiosc.2009.02.004
DO - 10.1016/j.jbiosc.2009.02.004
M3 - Article
VL - 107
SP - 662
EP - 667
JO - Journal of Bioscience and Bioengineering
JF - Journal of Bioscience and Bioengineering
SN - 1389-1723
IS - 6
ER -