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 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.

Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria

“…The sensitivity of the technique allows as little as 0?05 ng of total RNA to be detected, but the quantification of proportions is limited by the dynamic range of measurement. In practice, it is feasible to measure 100-200-fold differences in the amounts of individual target RNAs (Satokari et al, 2005). According to our results, the studied clostridial groups [Clostridium.…”

“…Faecal samples from the 32 subjects were obtained on two occasions 6 months apart (0 and 6 months). More detailed information about the IBS and control subject groups, as well as sampling and sample handling, has been described previously by Mättö et al Satokari et al (2005). Specific 16S rRNA-targeted probes for different groups of clostridia and a universal bacterial probe were used in the study ( Table 1).…”

“…RNA was extracted from faeces as described elsewhere (Zoetendal et al, 1998) with minor modifications (Satokari et al, 2005), and further purified by using the clean-up protocol of the RNeasy mini kit (Qiagen). Total RNA was quantified by A 260 measurement, and the purity of RNA was evaluated by agarose gel electrophoresis.…”

“…Purified RNA used for quantitative analysis (see below) was biotinylated by using Photoprobe biotin (Vector Laboratories). Biotinylation was performed by exposing the RNA to long-wave UV light (365 nm) for 30 min, and the subsequent purification of the RNA from free biotin was performed according to the manufacturer's instructions, with slight modifications according to Satokari et al (2005).…”

“…In the present study, the recently developed TRAC technique (Satokari et al, 2005) allowed the multiplexed quantification of the clostridial groups. The sensitivity of the technique allows as little as 0?05 ng of total RNA to be detected, but the quantification of proportions is limited by the dynamic range of measurement.…”