In situ detection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS

ABSTRACT An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes.

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Yn-situ: a robust single RNA molecule in situ detection method

10.1101/2021.10.20.465061 ◽

2021 ◽

Author(s):

Yunming Wu ◽

Wenjing Xu ◽

Limei Ma ◽

Zulin Yu ◽

Yongfu Wang ◽

...

Keyword(s):

Dynamic Range ◽

Signal To Noise Ratio ◽

High Sensitivity ◽

Cost Effective ◽

Hybridization Chain Reaction ◽

Single Pair ◽

Rna Molecules ◽

Key Steps ◽

In Situ Detection

We describe a cost-effective, highly sensitive, and quantitative method for in situ detection of single RNA molecules in tissue sections. This method, dubbed Yn situ, standing for Y-branched probe in situ hybridization, uses a single-strand DNA preamplifier with multiple initiation sites that trigger hybridization chain reaction (HCR) to detect polynucleotide. We characterized the performance of this method and compared it to other approaches in the postnatal mouse olfactory epithelia. We find that the Yn situ method, in conjunction with an improved fixation step, is sensitive enough to allow detection of single molecules using a single pair of probes targeting a short nucleotide sequence. A set of 5-probes can produce quantitative results with smaller puncta and higher signal-to-noise ratio than the 20-probe sets commonly required for HCR and RNA-Scope. We show that the high sensitivity and wide dynamic range allow quantification of genes expressed at different levels in the olfactory sensory neurons. We describe key steps of this method to enable broad utility by individual laboratories.

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Tissue-based in situ detection of the clarithromycin resistance for the personalized Helicobacter pylori eradication therapy

10.26226/morressier.596dfd5ad462b8029238774d ◽

2017 ◽

Author(s):

Éva Kocsmár

Keyword(s):

Helicobacter Pylori ◽

Eradication Therapy ◽

Helicobacter Pylori Eradication ◽

Clarithromycin Resistance ◽

In Situ Detection ◽

Helicobacter Pylori Eradication Therapy

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Rapid quantification and in situ detection of nitrifying bacteria in biofilms by monoclonal antibody method

Water Science & Technology ◽

10.2166/wst.2000.0459 ◽

2000 ◽

Vol 41 (4-5) ◽

pp. 301-308 ◽

Cited By ~ 1

Author(s):

N. Noda ◽

H. Ikuta ◽

Y. Ebie ◽

A. Hirata ◽

S. Tsuneda ◽

...

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Fluorescent Antibody ◽

Nitrifying Bacteria ◽

Fluorescent Antibody Technique ◽

Antibody Technique ◽

Antibody Method ◽

In Situ Detection ◽

Rapid Quantification

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

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Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

Journal of Clinical Medicine ◽

10.3390/jcm10020319 ◽

2021 ◽

Vol 10 (2) ◽

pp. 319

Author(s):

Hee Cheol Yang ◽

Won Jong Rhee

Keyword(s):

Extracellular Vesicles ◽

Liquid Biopsy ◽

Molecular Beacon ◽

Disease Diagnosis ◽

Single Step ◽

Flow Cytometer ◽

Surface Proteins ◽

Biomarker Detection ◽

In Situ Detection

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer.

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