Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture

Reetta Satokari (Corresponding Author), Kari Kataja, Hans Söderlund

Research output: Contribution to journalArticleScientificpeer-review

22 Citations (Scopus)

Abstract

Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin–nucleic acid–probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA–specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 102. The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA.
Original languageEnglish
Pages (from-to)120 - 127
Number of pages8
JournalMicrobial Ecology
Volume50
Issue number1
DOIs
Publication statusPublished - 2005
MoE publication typeA1 Journal article-refereed

Fingerprint

oligonucleotide probes
nucleic acids
nucleic acid
hybridization
probe
ribosomal RNA
quantitative analysis
streptavidin
fluorescent dyes
capillary electrophoresis
application methods
detection limit
feces
RNA
microorganisms
electrokinesis
microorganism
organisms
methodology
method

Keywords

  • nucleic acid sequences
  • biotinylated nucleic acid targets
  • oligonucleotide probes
  • methods

Cite this

Satokari, Reetta ; Kataja, Kari ; Söderlund, Hans. / Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture. In: Microbial Ecology. 2005 ; Vol. 50, No. 1. pp. 120 - 127.
@article{a1bb1033d7a74aa0a9e93454eac2fcf0,
title = "Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture",
abstract = "Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin–nucleic acid–probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA–specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 102. The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA.",
keywords = "nucleic acid sequences, biotinylated nucleic acid targets, oligonucleotide probes, methods",
author = "Reetta Satokari and Kari Kataja and Hans S{\"o}derlund",
year = "2005",
doi = "10.1007/s00248-004-0136-1",
language = "English",
volume = "50",
pages = "120 -- 127",
journal = "Microbial Ecology",
issn = "0095-3628",
publisher = "Springer",
number = "1",

}

Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture. / Satokari, Reetta (Corresponding Author); Kataja, Kari; Söderlund, Hans.

In: Microbial Ecology, Vol. 50, No. 1, 2005, p. 120 - 127.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture

AU - Satokari, Reetta

AU - Kataja, Kari

AU - Söderlund, Hans

PY - 2005

Y1 - 2005

N2 - Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin–nucleic acid–probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA–specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 102. The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA.

AB - Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin–nucleic acid–probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA–specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 102. The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA.

KW - nucleic acid sequences

KW - biotinylated nucleic acid targets

KW - oligonucleotide probes

KW - methods

U2 - 10.1007/s00248-004-0136-1

DO - 10.1007/s00248-004-0136-1

M3 - Article

VL - 50

SP - 120

EP - 127

JO - Microbial Ecology

JF - Microbial Ecology

SN - 0095-3628

IS - 1

ER -