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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
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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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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Single-Molecule Fluorescent In Situ Hybridization (smFISH) for RNA Detection in Bacteria.

Camilla Ciolli Mattioli1, Roi Avraham2

  • 1Department of Immunology and Regenerative Biology, Weizmann Institute of Science, Rehovot, Israel. Camilla.ciolli-mattioli@weizmann.ac.il.

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|March 19, 2024
PubMed
Summary

This chapter details single-molecule fluorescence in situ hybridization (smFISH) for bacterial mRNA quantification. The adaptable method offers insights into bacterial cell life and is easily adoptable by other labs.

Keywords:
BacteriasmFISH

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Bacterial gene expression regulation is complex.
  • Visualizing and quantifying mRNA in bacteria is crucial for understanding cellular processes.
  • Existing methods may have limitations in adaptability or accessibility.

Purpose of the Study:

  • To provide a detailed protocol for performing single-molecule fluorescence in situ hybridization (smFISH) in bacterial cells.
  • To outline methods for accurate quantification of mRNA transcripts using smFISH.
  • To highlight the adaptability and potential of smFISH for diverse bacterial research.

Main Methods:

  • Detailed step-by-step protocol for smFISH experiments in bacteria.
  • Techniques for imaging and analyzing fluorescently labeled mRNA molecules.
  • Strategies for quantifying mRNA transcript numbers per cell.

Main Results:

  • Successful implementation of smFISH for bacterial mRNA visualization.
  • Demonstration of accurate mRNA quantification across different bacterial species.
  • Validation of the method's flexibility under various experimental conditions.

Conclusions:

  • The described smFISH protocol is a robust and adaptable technique for bacterial mRNA analysis.
  • This method facilitates deeper insights into bacterial gene expression and cellular dynamics.
  • The approach is readily transferable to other laboratories, promoting wider adoption.