9.5: In Situ Hybridization

Search Fundamentals of Biochemistry

Learning Goals

• Define ISH and Distinguish Its Purpose:
  • Explain the principle of in situ hybridization, including how labeled complementary nucleic acid probes (DNA or RNA) are used to localize specific DNA or RNA sequences within fixed cells, tissues, or whole organisms.

• Differentiate ISH from Related Techniques:
  • Compare ISH with immunohistochemistry, highlighting how ISH detects nucleic acids while immunohistochemistry targets proteins.

• Describe Probe Design and Hybridization Conditions:
  • Identify the types of probes used in ISH (e.g., oligonucleotide probes, riboprobes) and explain how factors like temperature, salt concentration, and detergents are optimized to achieve high-stringency hybridization.

• Understand Signal Detection and Quantification:
  • Discuss various detection methods (autoradiography, fluorescence microscopy, immunohistochemical detection of antigen-labeled probes) and how signal intensity is used to infer the abundance and localization of target sequences.

• Differentiate ISH Applications:
  • Explain how DNA ISH is applied for chromosomal mapping and diagnostics (e.g., FISH for assessing chromosomal integrity) versus RNA ISH used for gene expression profiling (mRNA, lncRNA, miRNA localization) in various sample types, including circulating tumor cells.

• Evaluate Alternative ISH Technologies:
  • Describe the principles behind branched DNA (bDNA) assays for RNA detection, including their use of probe design and signal amplification to achieve single-molecule sensitivity and multiplexing capabilities.

• Interpret Experimental Data and Controls:
  • Discuss how multiplexing with multiple probes and the use of control probes (e.g., RNA spike-ins) help validate ISH experiments and ensure specificity and sensitivity.

These learning goals will help you integrate the theoretical principles of in situ hybridization with its practical applications in gene localization and expression analysis, deepening your understanding of how these techniques are employed in modern research and diagnostics.

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA, or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ) or if the tissue is small enough (e.g., plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH), in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.

In situ hybridization is used to reveal the location of specific nucleic acid sequences on chromosomes or in tissues, a crucial step for understanding genes' organization, regulation, and function. The key techniques currently in use include in situ hybridization to mRNA with oligonucleotide and RNA probes (both radio-labeled and hapten-labeled), analysis with light and electron microscopes, whole mount in situ hybridization, double detection of RNAs and RNA plus protein, and fluorescent in situ hybridization to detect chromosomal sequences. DNA ISH can be used to determine the structure of chromosomes. For example, fluorescent DNA ISH (FISH) can be used in medical diagnostics to assess chromosomal integrity. RNA ISH (RNA in situ hybridization) is used to measure and localize RNAs (mRNAs, lncRNAs, and miRNAs) within tissue sections, cells, whole mounts, and circulating tumor cells (CTCs). In situ hybridization was invented by Mary-Lou Pardue and Joseph G. Gall.

Figure 5.28 In Situ Hybridization of wild-type Drosophila embryos at different developmental stages for the RNA from a gene called hunchback. Image by Nina

For hybridization histochemistry, sample cells and tissues are usually treated to fix the target transcripts and increase access to the probe. As noted above, the probe is either a labeled complementary DNA or, now most commonly, a complementary RNA (riboprobe). The probe hybridizes to the target sequence at elevated temperature. Then, the excess probe is washed away (after prior hydrolysis using RNase in the case of an unhybridized, excess RNA probe). Solution parameters such as temperature, salt, and/or detergent concentration can be manipulated to remove non-identical interactions (i.e., only exact sequence matches will remain bound). Then, the probe labeled with either radio-, fluorescent- or antigen-labeled bases (e.g., digoxigenin) is localized and quantified in the tissue, using either autoradiography, fluorescence microscopy, or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactivity or other non-radioactive labels to detect two or more transcripts simultaneously.

An alternative technology, branched DNA assay, can be used for RNA (mRNA, lncRNA, and miRNA) in situ hybridization assays with single molecule sensitivity without radioactivity. This approach (e.g., ViewRNA assays) can be used to visualize up to four targets in one assay, and it uses patented probe design and bDNA signal amplification to generate sensitive and specific signals. Samples (cells, tissues, and CTCs) are fixed and treated to allow RNA target accessibility (RNA un-masking). Target-specific probes hybridize to each target RNA. Subsequent signal amplification is predicated by specific hybridization of adjacent probes (individual oligonucleotides [oligos] that bind side by side on RNA targets). A typical target-specific probe will contain 40 oligonucleotides, resulting in 20 oligo pairs that bind side-by-side on the target to detect mRNA and lncRNA, and 2 oligos or a single pair for miRNA detection. Signal amplification is achieved via a series of sequential hybridization steps. A pre-amplifier molecule hybridizes to each oligo pair on the target-specific RNA, and then multiple amplifier molecules hybridize to each pre-amplifier. Next, multiple-label probe oligonucleotides (conjugated to alkaline phosphatase or directly to fluorophores) hybridize to each amplifier molecule. A fully assembled signal amplification structure, “Tree,” has 400 binding sites for the label probes. When all target-specific probes bind to the target mRNA transcript, an 8,000-fold signal amplification occurs for that one transcript. Separate but compatible signal amplification systems enable multiplex assays. The signal can be visualized using a fluorescence or brightfield microscope.

Summary

This chapter provides a comprehensive overview of in situ hybridization (ISH), a powerful technique for localizing specific nucleic acid sequences directly within cells, tissues, or even whole organisms. Designed for junior and senior biochemistry majors, the chapter begins by defining ISH and contrasting it with related methods such as immunohistochemistry, which localizes proteins rather than nucleic acids.

At the core of ISH is the use of labeled complementary probes—either DNA, RNA, or modified nucleic acids—that bind to target sequences (e.g., mRNA, lncRNA, miRNA, or specific DNA regions) under optimized hybridization conditions. These conditions, which include controlled temperature, salt, and detergent levels, ensure that only specific, high-affinity interactions are maintained, while non-specific binding is minimized through stringent washes.

The chapter reviews the various formats of ISH, including traditional section-based ISH, whole mount ISH (for small tissues like Drosophila embryos), and applications to circulating tumor cells. It also discusses different labeling strategies such as radioactive, fluorescent, and antigen-based (e.g., digoxigenin) labels, and the detection methods employed—ranging from autoradiography and fluorescence microscopy to immunohistochemical detection—to visualize and quantify probe-target hybridization.

A special emphasis is placed on advanced ISH technologies like branched DNA (bDNA) assays. These assays utilize a series of sequential hybridization and signal amplification steps, enabling the detection of individual RNA molecules with high sensitivity and allowing for the simultaneous multiplex detection of multiple targets.

In summary, this chapter ties together the principles of nucleic acid hybridization with practical applications in gene mapping, expression profiling, and diagnostics. Through an exploration of probe design, hybridization conditions, and detection strategies, students gain insight into how ISH is used to elucidate the spatial organization and regulation of genetic information in both health and disease.