How to Use Fluorescence In Situ Hybridization
Introduction
Fluorescence In Situ Hybridization, commonly known as FISH, is a powerful molecular biology technique that allows scientists to visualize and analyze the presence and location of specific nucleic acid sequences within cells or tissues. Developed in the 1980s, FISH has become an indispensable tool in various scientific disciplines, including genetics, cytogenetics, and cancer research. In this comprehensive guide, we will explore the principles, applications, and step-by-step procedures involved in using Fluorescence In Situ Hybridization.
Figure 1. Fluorescence in situ hybridization for trisomy 12. (Shakoori AR. 2017)
Principles of Fluorescence In Situ Hybridization
FISH relies on the principle of hybridization, where a fluorescently labeled DNA or RNA probe is used to bind specifically to its complementary target sequence in the sample. The process involves several key steps:
Probe Design: The first step in FISH is designing a probe that is complementary to the target nucleic acid sequence of interest. Probes are typically labeled with fluorescent dyes, allowing for easy visualization under a fluorescence microscope.
Denaturation: The sample, usually fixed cells or tissue sections, is treated to denature or separate the DNA strands. This step ensures accessibility of the target sequences for hybridization.
Hybridization: The labeled probe is then applied to the sample, allowing it to hybridize or bind specifically to its complementary sequence within the denatured DNA. This step is crucial for the specificity and success of the FISH assay.
Washing: Excess, unbound probes are removed through a series of washing steps, enhancing the signal-to-noise ratio and reducing background fluorescence.
Detection: The final step involves detecting the fluorescent signals emitted by the labeled probes under a fluorescence microscope. The signals can be captured and analyzed to determine the presence, location, and abundance of the target sequences.
Applications of FISH
FISH has a wide range of applications in various scientific fields:
Cytogenetics: FISH is extensively used in cytogenetics to study the structure and behavior of chromosomes. It can identify chromosomal abnormalities, such as translocations, deletions, and duplications, providing valuable insights into genetic disorders and cancer.
Cancer Research: FISH is a valuable tool for studying cancer genetics. It can help identify specific genetic alterations associated with different types of cancer, aiding in diagnosis, prognosis, and treatment decisions.
Microbial Ecology: In environmental microbiology, FISH is employed to identify and quantify specific microorganisms in environmental samples. This allows researchers to study microbial communities and their dynamics in different ecosystems.
Preimplantation Genetic Diagnosis (PGD): FISH has been used in PGD to screen embryos for chromosomal abnormalities before implantation during in vitro fertilization (IVF) procedures, reducing the risk of genetic disorders in offspring.
Neuroscience: FISH is utilized in neuroscience to study gene expression patterns in the brain. It helps researchers understand the molecular mechanisms underlying neurological disorders and development.
Step-by-Step Procedure for Performing FISH
Performing FISH involves a series of carefully orchestrated steps to ensure accurate and reliable results. Here is a step-by-step guide:
Sample Preparation:
Fixation: Fix the cells or tissue samples using an appropriate fixative to preserve cellular structures and prevent degradation.
Permeabilization: Treat the fixed samples with a permeabilization agent to allow the probe to penetrate the cell membranes and access the target DNA.
Probe Preparation:
Labeling: Fluorescently label the DNA or RNA probe using a suitable labeling method. Common labels include fluorophores like fluorescein isothiocyanate (FITC) or rhodamine.
Denaturation: Denature the labeled probe to ensure that it remains single-stranded and can hybridize with the target sequences.
Hybridization:
Application: Apply the denatured, labeled probe to the prepared sample, ensuring even distribution.
Incubation: Incubate the sample at an optimal temperature to allow for the specific hybridization of the probe with its complementary target sequences.
Washing:
Stringency Washes: Conduct a series of washing steps with solutions of varying stringency to remove excess, unbound probes and reduce background fluorescence.
Detection:
Mounting: Mount the sample onto a microscope slide, using an appropriate mounting medium to preserve the fluorescence signals.
Microscopy: Examine the sample under a fluorescence microscope equipped with suitable filters for the fluorophores used. Capture images of the fluorescent signals.
Analysis: Analyze the images to determine the presence, location, and intensity of the fluorescent signals, providing insights into the distribution of the target sequences.
Troubleshooting Tips
Background Signal:
Increase stringency during washing steps.
Use blocking agents to reduce non-specific binding.
Weak Signal:
Optimize probe concentration.
Ensure proper denaturation and hybridization conditions.
High Background Fluorescence:
Improve permeabilization to enhance probe penetration.
Check the quality of reagents, especially the labeled probe.
Uneven Signal Distribution:
Ensure even probe distribution during application.
Use appropriate mounting techniques for uniform sample presentation.
Conclusion
Fluorescence In Situ Hybridization is a versatile and indispensable technique with applications spanning genetics, cytogenetics, cancer research, microbiology, and neuroscience. Understanding the principles and following a well-optimized protocol is crucial for obtaining reliable and meaningful results. As technology advances, FISH continues to play a pivotal role in expanding our understanding of the molecular intricacies within cells and tissues, contributing to advancements in both basic research and clinical applications.
Related Products/Services
Fluorescence In Situ Hybridization
- Shakoori AR. Fluorescence In Situ Hybridization (FISH) and Its Applications. Chromosome Structure and Aberrations. 2017, 10:343–67.
*If your organization requires the signing of a confidentiality agreement, please contact us by email.
Please note: Our services can only be used for research purposes. Do not use in diagnostic or therapeutic procedures!