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

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hirokazu Takahashi ◽  
Kyohei Horio ◽  
Setsu Kato ◽  
Toshiro Kobori ◽  
Kenshi Watanabe ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Mrna Expression ◽  
Direct Detection ◽  
Rolling Circle Amplification ◽  
Rnase H ◽  
Microbial Cells ◽  
Rolling Circle

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

2019 ◽  
Author(s):  
Hirokazu Takahashi ◽  
Kyohei Horio ◽  
Setsu Kato ◽  
Toshiro Kobori ◽  
Kenshi Watanabe ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Direct Detection ◽  
Rolling Circle Amplification ◽  
Rnase H ◽  
Microbial Cells ◽  
Rolling Circle ◽  
The Individual ◽  
Meta Analyses

ABSTRACTMeta-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.

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

2021 ◽  
Vol 9 (6) ◽  
pp. 1208
Author(s):  
Kyohei Horio ◽  
Hirokazu Takahashi ◽  
Toshiro Kobori ◽  
Kenshi Watanabe ◽  
Tsunehiro Aki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lactic Acid ◽  
In Situ Hybridization ◽  
Lactic Acid Bacteria ◽  
Fluorescence In Situ Hybridization ◽  
Rolling Circle Amplification ◽  
Rnase H ◽  
Microbial Interactions ◽  
Rolling Circle

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 Legionellae in Respiratory Tract Specimens by Using Fluorescence In Situ Hybridization

Legionella ◽  
2014 ◽  
pp. 221-224
Author(s):  
Jinxin Hu ◽  
Ajit P. Limaye ◽  
Matthias Horn ◽  
Stefan Juretschko ◽  
Romesh Gautom ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Respiratory Tract ◽  
Fluorescence In Situ Hybridization ◽  
Direct Detection

scholarly journals Organization of small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in situ hybridization and immunocytochemistry.

1994 ◽  
Vol 5 (12) ◽  
pp. 1289-1299 ◽  
Author(s):  
A G Matera ◽  
K T Tycowski ◽  
J A Steitz ◽  
D C Ward
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Rnase H ◽  
Rrna Processing ◽  
Pol I ◽  
Rrna Maturation ◽  
Polymerase I ◽  
Processing Factors

The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations.

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