Visualization of Gene Reciprocity among Lactic Acid Bacteria in Yogurt by RNase H-Assisted Rolling Circle Amplification-Fluorescence In Situ Hybridization.

Recently, we developed an in situ mRNA detection method termed RNase H-assisted rolling circle amplification-fluorescence in situ hybridization (RHa-RCA-FISH), which can detect even short mRNA in a bacterial cell. However, because this FISH method is sensitive to the sample condition, it is necessary to find a suitable cell permeabilization and collection protocol. Here, we demonstrate its further applicability for detecting intrinsic mRNA expression using lactic acid bacteria (LAB) as a model consortium. Our results show that this method can visualize functional gene expression in LAB cells and can be used for monitoring the temporal transition of gene expression. In addition, we also confirmed that data obtained from bulk analyses such as RNA-seq or microarray do not always correspond to gene expression in individual cells. RHa-RCA-FISH will be a powerful tool to compensate for insufficient data from metatranscriptome analyses while clarifying the carriers of function in microbial consortia. By extending this technique to capture spatiotemporal microbial gene expression at the single-cell level, it will be able to characterize microbial interactions in phytoplankton-bacteria interactions.

Direct detection of mRNA expression in microbial cells by fluorescence in situ hybridization using RNase H-assisted rolling circle amplification.

Meta-analyses using next generation sequencing is a powerful strategy for studying microbiota; however, it cannot clarify the role of individual microbes within microbiota. To know which cell expresses what gene is important for elucidation of the individual cell's function in microbiota. In this report, we developed novel fluorescence in situ hybridization (FISH) procedure using RNase-H-assisted rolling circle amplification to visualize mRNA of interest in microbial cells without reverse transcription. Our results show that this method is applicable to both Gram-negative and Gram-positive microbes without any noise from DNA, and it is possible to visualize the target mRNA expression directly at the single-cell level. Therefore, our procedure, when combined with data of meta-analyses, can help to understand the role of individual microbes in the microbiota.

RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection.

RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3'-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.