Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture.
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 10(2). 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.
Satokari
Reetta M
RM
VTT Biotechnology, Espoo, Finland. reetta.satokari@vtt.fi
Kataja
Kari
K
Söderlund
Hans
H
D001419:Bacteria / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D017403:In Situ Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S / Q000032:analysis* / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S