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In-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 labe…
In situ hybridization is a technique used to locate DNA or RNA in cultured cells or in chemically preserved tissue sections.
RNA in situ hybridization is used to monitor where a gene is being expressed by detecting and quantifying the specific mRNA transcripts.
The target mRNAs are detected by single-stranded, complementary RNA probes that are created by in vitro transcription of a DNA template or synthesized by a chemical reaction.
The probes are labeled with radioactive isotopes, fluorophores, or other reporter molecules, such as biotin or digoxigenin, that can be bound and detected by a labeled antibody.
RNA in situ hybridization has four main steps: tissue fixation, permeabilization, hybridization, and detection.
To begin, fresh tissue is harvested and chemically treated to stop any biochemical reactions within the tissue and preserve its structural integrity. This preservation technique is called tissue fixation.
Next, proteins and lipids in the tissue need to be removed for the probe to access and bind the target RNA.
To degrade the proteins and permeabilize the cell membrane, the sample is treated with 0.2 molar hydrochloric acid and enzymes such as proteinases, while detergents are used to break down the lipids.
The target RNA is then hybridized with the probes at a temperature just below the melting point of the RNA-probe hybrid. This helps the probes to anneal with the complementary mRNAs. Unbound and loosely bound probes are removed by performing several washes.
The method for detecting the hybridized probes depends on the type of label. Radioactive probes are exposed to photographic films, while fluorescent probes are visible under fluorescent microscopes.
For other labels, antibodies against the reporter molecule are tagged with a fluorescent or colorimetric dye.
Detection of hybridized probes reveals the distribution of specific mRNAs indicating where the genes of interest are expressed.
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Q1: What is in situ hybridization used for in molecular biology?
In situ hybridization is a technique that detects and localizes specific DNA or RNA molecules in cultured cells or preserved tissue sections. RNA in situ hybridization specifically monitors where genes are expressed by detecting and quantifying mRNA transcripts using complementary RNA probes labeled with radioactive isotopes, fluorophores, biotin, or digoxigenin for visualization.
Q2: What are the four main steps of RNA in situ hybridization?
The four main steps are tissue fixation, permeabilization, hybridization, and detection. Tissue fixation chemically preserves structural integrity. Permeabilization removes proteins and lipids using hydrochloric acid, proteinases, and detergents to allow probe access. Hybridization anneals probes to complementary mRNAs at controlled temperatures. Detection reveals mRNA distribution using methods suited to the probe label type.
Q3: How do different probe labels affect detection in in situ hybridization?
Detection methods depend on the probe label type. Radioactive probes are exposed to photographic films via autoradiography. Fluorescent probes are visualized under fluorescent microscopes. For other labels like biotin or digoxigenin, antibodies tagged with fluorescent or colorimetric dyes bind the reporter molecule for visualization, enabling flexible detection strategies.
Q4: What types of probes are used in in situ hybridization?
Multiple probe types are employed, including single-stranded DNA probes, double-stranded DNA probes, antisense RNA probes (riboprobes), and synthetic oligodeoxynucleotide probes. Probe selection depends on sensitivity, specificity, stability, and tissue penetration ability. These probes are complementary strands that bind to corresponding nucleotide sequences in cells or tissue samples.
Q5: Why is tissue permeabilization necessary in in situ hybridization?
Tissue permeabilization removes proteins and lipids that block probe access to target RNA. The sample is treated with hydrochloric acid and proteinases to degrade proteins and permeabilize cell membranes, while detergents break down lipids. This step ensures probes can penetrate tissue and successfully anneal with complementary mRNA sequences.
Q6: What are the advantages of in situ hybridization for gene expression analysis?
In situ hybridization can be applied to frozen tissues, maximizing use of difficult-to-obtain samples. It can be combined with other techniques like immunohistochemistry to detect both protein and active mRNA simultaneously. However, samples with low DNA and RNA copy numbers may present challenges for target identification.
Q7: How are probes labeled and detected in in situ hybridization?
Probes are labeled using radioisotopes (3H, 35S, 32P), non-radioactive labels (biotin, digoxigenin, fluorescein), or fluorescent dyes attached via end-labeling, nick-translation, or random primer synthesis. These labeled probes enable detection through reporter genes or antibodies tagged with fluorescent or colorimetric dyes, allowing visualization of hybridized target sequences.