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Related Concept Videos

In-situ Hybridization02:31

In-situ Hybridization

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In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...
9.7K
FISH - Fluorescent In-situ Hybridization02:07

FISH - Fluorescent In-situ Hybridization

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Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...
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Localization of Long Noncoding RNA in Formalin-Fixed, Paraffin-Embedded Vascular Tissue Using In Situ Hybridization.

Jessica P Scanlon1, Andrew H Baker1,2, Judith C Sluimer3,4

  • 1BHF Centre for Cardiovascular Sciences (CVS), University of Edinburgh, Edinburgh, UK.

Methods in Molecular Biology (Clifton, N.J.)
|March 3, 2022
PubMed
Summary

In situ hybridization (ISH) detects RNA location in tissues. This method is crucial for localizing long noncoding RNAs (lncRNAs) in vascular tissues, especially when RNA abundance is low.

Keywords:
In situ hybridizationLocalizationLong noncoding RNAVascular

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • In situ hybridization (ISH) is a key technique for visualizing RNA within its native tissue context.
  • Long noncoding RNAs (lncRNAs) play critical roles but require specific methods for localization due to low abundance and lack of protein products.
  • Accurate tissue and cellular localization of lncRNAs is essential for understanding their function.

Purpose of the Study:

  • To detail the methodology and theoretical underpinnings of performing ISH for lncRNA detection.
  • To provide practical guidance for applying ISH to lncRNA analysis in vascular tissues.
  • To highlight strategies for enhancing detection sensitivity for low-abundance lncRNAs.

Main Methods:

  • Utilizing oligonucleotide probes with complementary sequences to target specific RNA molecules.
  • Employing colorimetric detection systems for the visualization of probe-target binding.
  • Implementing double-digoxigenin (DIG) labeling to amplify signal for low-abundance targets.

Main Results:

  • Demonstrated the capability of ISH to precisely determine the location of lncRNA within specific cell types and intracellular compartments.
  • Successfully applied ISH for lncRNA detection in vascular tissues, confirming its utility in this context.
  • Showcased enhanced detection sensitivity through double-DIG labeling, crucial for scarce RNA species.

Conclusions:

  • ISH is an indispensable technique for the spatial and cellular localization of lncRNAs.
  • The described ISH protocol, particularly with enhanced DIG labeling, is effective for studying lncRNAs in vascular tissues.
  • This approach facilitates a deeper understanding of lncRNA function through precise tissue-level analysis.