Toehold-initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells

2014 ◽  
Vol 126 (9) ◽  
pp. 2421-2425 ◽  
Author(s):  
Ruijie Deng ◽  
Longhua Tang ◽  
Qianqian Tian ◽  
Ying Wang ◽  
Lei Lin ◽  
...  
Keyword(s):  
Single Cells ◽  
Rolling Circle Amplification ◽  
Rolling Circle

Toehold-initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells

2014 ◽  
Vol 53 (9) ◽  
pp. 2389-2393 ◽  
Author(s):  
Ruijie Deng ◽  
Longhua Tang ◽  
Qianqian Tian ◽  
Ying Wang ◽  
Lei Lin ◽  
...  
Keyword(s):  
Single Cells ◽  
Rolling Circle Amplification ◽  
Rolling Circle

scholarly journals Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification

2017 ◽  
Vol 8 (5) ◽  
pp. 3668-3675 ◽  
Author(s):  
Ruijie Deng ◽  
Kaixiang Zhang ◽  
Yupeng Sun ◽  
Xiaojun Ren ◽  
Jinghong Li
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Rolling Circle Amplification ◽  
Robust Method ◽  
Single Nucleotide ◽  
Rolling Circle ◽  
Target Rna

We report a robust method for the efficient imaging of mRNA with single-nucleotide and near-single-molecule resolution in single cells.

scholarly journals Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

2005 ◽  
Vol 71 (12) ◽  
pp. 7933-7940 ◽  
Author(s):  
Fumito Maruyama ◽  
Takehiko Kenzaka ◽  
Nobuyasu Yamaguchi ◽  
Katsuji Tani ◽  
Masao Nasu
Keyword(s):  
Gene Sequence ◽  
Rolling Circle Amplification ◽  
Specific Gene ◽  
Toxin Gene ◽  
Dna Uptake ◽  
Fluorescent Intensity ◽  
Prolonged Incubation ◽  
Rolling Circle ◽  
E Coli

ABSTRACT Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.

Lectin-mediated in situ rolling circle amplification on exosomes for probing cancer-related glycan pattern

2018 ◽  
Vol 1039 ◽  
pp. 108-115 ◽  
Author(s):  
Yimei Feng ◽  
Yuna Guo ◽  
Yiran Li ◽  
Jing Tao ◽  
Lin Ding ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Rolling Circle ◽  
Glycan Pattern

In Situ Detection of Messenger RNA Using Digoxigenin-Labeled Oligonucleotides and Rolling Circle Amplification

2001 ◽  
Vol 70 (3) ◽  
pp. 281-288 ◽  
Author(s):  
Yi Zhou ◽  
Margaret Calciano ◽  
Stefan Hamann ◽  
J.H. Leamon ◽  
Tod Strugnell ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Rolling Circle Amplification ◽  
Rolling Circle ◽  
In Situ Detection

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.

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