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Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos
Published on: January 30, 2013
In situ hybridization to cellular RNA
R Zeller1, M Rogers, A G Haramis
1University of Utrecht, The Netherlands.
Current Protocols in Molecular Biology
|February 12, 2008
Summary
This study details in situ hybridization protocols for locating cellular RNA in tissues. It covers both radioactive and non-radioactive probe synthesis and detection methods for precise message mapping.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- In situ hybridization is crucial for determining the cellular localization of specific RNA messages within complex cell populations and tissues.
- Accurate localization of RNA is essential for understanding gene expression and cellular function.
Purpose of the Study:
- To describe detailed protocols for in situ hybridization to cellular RNA.
- To provide methods for hybridizing slide-mounted paraffin sections or cryosections with labeled probes.
- To outline the synthesis and detection of both radioactive and non-radioactive RNA probes.
Main Methods:
- Protocols for hybridizing slide-mounted paraffin sections and cryosections with labeled probes.
- Synthesis of 35S-labeled riboprobes and double-stranded DNA (dsDNA) probes.
- Detection of probes using film or emulsion autoradiography.
- Synthesis of digoxigenin-labeled RNA probes for non-radioactive detection.
Main Results:
- Established protocols for effective in situ hybridization on tissue sections.
- Demonstrated the synthesis and application of both radioactive (35S) and non-radioactive (digoxigenin) probes.
- Highlighted the advantages of digoxigenin-labeled RNA probes, including ease of synthesis, stability, reusability, and cleaner signals due to ribonuclease treatment.
Conclusions:
- The described methods provide a comprehensive toolkit for researchers to perform in situ hybridization.
- Non-radioactive digoxigenin-labeled RNA probes offer significant advantages over traditional radioactive probes for cellular RNA localization.
- These protocols facilitate precise mapping of RNA messages in diverse biological samples.
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,...
02:57Southern Blot
Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
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