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12:31
Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization
Published on: March 25, 2009
High-resolution whole-mount in situ hybridization using Quantum Dot nanocrystals
Andriani Ioannou1, Iro Eleftheriou, Andrea Lubatti
1Department of Biological Sciences, University of Cyprus, 1678 Nicosia, Cyprus.
Journal of Biomedicine & Biotechnology
|January 31, 2012
Summary
Quantum dots (QDs) offer superior sensitivity and photostability for whole-mount in situ hybridization (WISH) in embryos. This method enables clear visualization of mRNA transcripts without enzymatic amplification, improving resolution and imaging capabilities.
Area of Science:
- Biotechnology
- Molecular Biology
- Developmental Biology
Background:
- Whole-mount fluorescent in situ hybridization (WISH) is crucial for detecting mRNA in intact embryos.
- Traditional organic fluorophores used in WISH suffer from photobleaching and limited brightness.
- Nanoparticle-based probes are needed to enhance sensitivity and resolution in WISH.
Purpose of the Study:
- To develop a direct quantum dot (QD) labeling method for oligonucleotide probes in WISH.
- To improve sensitivity, resolution, and photostability in WISH detection of mRNA transcripts.
- To enable advanced imaging of mRNA expression patterns in 3D.
Main Methods:
- Direct QD labeling of modified oligonucleotide probes via streptavidin-biotin and antibody-mediated interactions.
- Treatment of embryos with proteinase K to enhance probe penetration.
- Utilizing QDs as fluorescent reporters for mRNA detection in whole embryos.
Main Results:
- QD labeling significantly increased WISH sensitivity compared to organic fluorophores.
- Enabled direct fluorescent detection of specific transcripts without enzymatic amplification.
- Overcame photostability and brightness limitations of organic fluorophores.
Conclusions:
- QD-based WISH offers enhanced sensitivity and resolution for mRNA detection in intact embryos.
- This method facilitates intracellular mRNA localization, multiplexed detection, and 3D expression pattern visualization.
- QDs provide a photostable and bright alternative for advanced whole-mount gene expression analysis.
Related Concept Videos
02:31In-situ Hybridization
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...
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...
02:07FISH - Fluorescent In-situ Hybridization
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,...