Chapter 3. In Situ Detection of Intracellular Messenger RNA and MicroRNA

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

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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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Asymmetric reconstruction of mammalian reovirus reveals interactions among RNA, transcriptional factor µ2 and capsid proteins

Nature Communications ◽

10.1038/s41467-021-24455-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Muchen Pan ◽

Ana L. Alvarez-Cabrera ◽

Joon S. Kang ◽

Lihua Wang ◽

Chunhai Fan ◽

...

Keyword(s):

Rna Polymerase ◽

Molecular Interactions ◽

Messenger Rna ◽

Rna Binding ◽

Capsid Proteins ◽

Rna Transcription ◽

Infectious Particle ◽

Virion Assembly ◽

Rna Capping

AbstractMammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor μ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase μ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D3d symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.

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