In Situ Hybridization Buyer’s Guide

In situ hybridization (ISH) is a powerful and versatile technique that is used to localize and detect the distribution of specific nucleic acid (DNA or RNA) sequences, relative to their protein products and other cellular components, within a heterogenous cell population. The visualization of precise spatial and temporal genomic loci and gene-expression change within a tissue, cell, or chromosome, makes ISH a very useful tool for biological and diagnostic applications. ISH, also referred to as hybridization histochemistry, was originally developed by Pardue and Gall¹ and independently by John et al.² in 1969.

Traditionally, nucleic acid detection using blot hybridization involves isolation of DNA or RNA sequences from the sample by separating it on a gel, blotting it onto nitrocellulose, and probing it with a complementary DNA or RNA sequence (probe). In the case of ISH, the principle remains the same except that sequences are now directly detected within a cell or tissue sample (hence the name in situ). ISH can be used on a variety of different samples such as morphologically preserved chromosomes, cells, tissue sections, entire tissue (whole mount ISH), and circulating tumor cells (CTCs). It can be used to identify and roughly quantify specific cellular DNA, messenger RNA (mRNA), microRNA (miRNA), noncoding RNA, and other nucleotide entities, thereby offering insight into some of the earliest changes taking place in the cell.

Multiple hybridizations can be performed on the same sample or libraries of samples can be made and stored for future use. This becomes important when working with small amounts or hard-to-get samples from embryos, clinical biopsies, and autopsies. When compared to other nucleic acid assays like Southern/Northern blotting and PCR, ISH provides additional information on nucleic acid localization. This information, when combined with histopathological data, offers a complete picture of the cellular changes taking place.

What does ISH involve?

ISH is based on a property called annealing, where nucleic acids with sufficient complementarity bind to each other and form hybrids. Hybrids form between naturally occurring or artificially created complementary strands of DNA-DNA, DNA-RNA, or RNA-RNA sequences. Hence, ISH probes can be either double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA (riboprobes), or synthetic oligonucleotides. The hybrids can be visualized and detected using a label attached to the probe. The labels used are either radioactive isotopes (P³², S³⁵, H³) or nonradioactive molecules like biotin, digoxigenin, or fluorescent dyes. Nonradioactive hybridization methods can further be classified as direct or indirect.

In the direct method, a reporter molecule is attached to the probe, which is immediately visualized under a microscope after hybridization, using either fluorescence or chromogenic detection. In the indirect method, the probe contains a reporter molecule, which is detected once it binds to another label. Biotin is commonly used in the indirect method to label the probe. Biotin, in turn, binds to either avidin or streptavidin, which is conjugated to alkaline phosphatase or horseradish peroxidase. Digoxigenin is another probe label that can be detected by digoxigenin-specific antibodies. “Digoxigenin-based detection overcomes high background and false positives that may arise from endogenously expressed biotin in biological samples,” says Morgan Mathieu, Ph.D., senior application specialist at Enzo Life Sciences.

What are the different types of ISH?

Chromogenic in situ hybridization (CISH) uses peroxidase- or alkaline phosphatase-labeled reporter antibodies that hybridize with the DNA probe, and are detected with an enzymatic reaction using bright-field microscopy.³ With CISH, changes in gene amplification, gene deletion, chromosomal translocations, and chromosomal number can all be detected at the same time, along with tissue morphology. CISH has some advantages in that it uses low cost light microscopy and the staining of the sample is permanent.

Fluorescence in situ hybridization (FISH) uses directly labeled fluorescent nucleotides or probes with reporter molecules that are indirectly detected by fluorescent antibodies or affinity molecules.⁴ The probes bind complementary nucleotide sequences resulting in colored signals (fluorescent spots) that can be detected in situ using a fluorescent microscope. FISH is highly sensitive and can be used on fresh, frozen, or paraffin-embedded samples making it a versatile and useful technique for diagnostic applications. Spectrally distinct fluorophore labels can be used for each probe, making multiplexing and visualizing co-localization of nucleotides easier and better. FISH is commonly used for detection of structural rearrangements such as translocations, inversions, insertions, and microdeletions, as well as for chromosomal gene mapping and for identifying and characterizing chromosome breakpoints.

FISH has considerably bridged the gap between conventional cytogenetics and molecular biology, with respect to sample resolution, and the technique continues to evolve. Multicolor FISH can identify chromosomal alterations using two or more probes, each specifically labeled with different fluorescent colors. Types of multicolor FISH include multiplex-FISH, spectral karyotyping, cross-species color banding, and comparative genomic hybridization (CGH). Multiplex-FISH and spectral karyotyping provide the same type of information but have different ways of acquiring and processing images of chromosomes. Cross-species color banding is not as good for identifying translocations between different chromosomes, but it can be useful for identifying certain intra-chromosomal rearrangements.

CGH is a two-color quantitative FISH method mostly used for investigating copy number variations (CNV) in DNA from two different sources. Array CGH uses nucleic acid sequences derived from various sources immobilized on glass slides and can measure CNV with greater degree of resolution and multiplexing. CNV are linked to predisposition to many diseases and have tremendous potential for diagnostics.

A new technique called CASFISH uses the clustered regularly interspaced short palindromic repeats (CRISPR) and nuclease-deficient CRISPR-associated caspase 9 or dead Cas9 (dCas9) complex as a probe to label specific DNA sequences.⁵ CASFISH assays using fluorescently labeled dCas9 coupled with various single-guide RNA (sgRNA) complexes have been shown to be rapid, robust, and cost-effective for multiplexed labeling, in fixed and living cells, for both research and diagnostic applications.

What can ISH be used for?

Similar to other nucleic acid assays, ISH helps in identification of genes and gene expression, but it is unique in that it does not require removing the target nucleic acid sequence from its topographical surroundings, and hence can be used for localization as well. Whole genome, entire chromosome, chromosomal-specific regions, or single-copy sequences can be identified using ISH, depending on the probe used. This results in many diverse applications for ISH in research, clinical, and diagnostic settings.

DNA ISH can be used to determine the structure of chromosomes and assess chromosomal integrity. RNA ISH can be used to measure and localize different types of RNAs (mRNAs, lncRNAs, noncoding RNAs, and miRNAs) within a variety of samples. “Although RNA probes can be very problematic to work with, RNA ISH has been gaining traction in the last ten years or so,” says Mathieu.

“RNA-RNA hybrids tend to be thermostable and resistant to RNase activity. RNases can be used to eliminate nonhybridized RNA thereby reducing unspecific staining.” FISH has been used in genomics, cell biology, epigenetics, prenatal research, oncology, neuroscience, toxicology, microbiology, developmental biology, pathology, and clinical research. Diagnostic tests using FISH have been used for many genetic diseases such as leukemia, lymphoma, solid tumors, autism, infectious diseases, and other developmental syndromes.

What are some of the improvements in ISH?

ISH involves many steps that need to be performed with precision, irrespective of the sample and probe that is used. From preserving the target sequence in the sample, which starts with cutting, fixing, staining, and freezing the cell or tissue appropriately, to detecting the hybridization signal, everything requires skill and the use of right equipment and reagents to avoid artifacts and errors. “Challenges can be encountered each step of the way,” says Mathieu. “To start, there are as many different protocols as there are tissue types and targets to be analyzed and probed. That is why it is absolutely essential to have a thorough understanding of the objective and of the technique itself, prior to undertaking this relatively complicated experiment.”

Until recently, ISH was limited by both availability and costs of the hardware, software, reagents, and probe technology.

Until recently, ISH was limited by both availability and costs of the hardware, software, reagents, and probe technology. Researchers had to customize their FISH systems using various reagents, probes, and microscopes, until the systems became commercially available. Microscope hardware optimized for multicolor FISH was not available until the mid-1990s. There were limitations to the image analysis software in terms of superimposing images of different colors and quantitatively analyzing samples, and researchers had to develop their own in-house software for data collection and analysis.

In the last couple of decades, incredible developments on various fronts have led to several improvements in ISH. Several good quality, directly labeled commercial probes that provide high resolution and fast hybridization are now available, thus making ISH accessible to clinical and diagnostic laboratories.

Richard S. Smith, Ph.D., application scientist-genomics at Agilent Technologies, explains the difference between their DNA FISH probes that are designed in silico and chemically synthesized versus FISH probes that are manufactured with bacterial artificial chromosome (BAC) technology. “An oligo probe consists of many short DNA oligos tiled along the region to be assessed, with the repeat regions omitted, to ensure robust signal targeted only to the region of interest,” says Smith. “Each BAC probe contains one long DNA sequence (100 Mb or longer) that includes the sequence over all the repeat regions, which can lead to lower resolution. Another difference is that oligo probes come ready to use, while some BAC-based probes require additional prep.”

According to Mathieu, DNA and RNA probes that are available commercially are expensive and do not always offer experimental flexibility as they come with a single label type. Offering labeling systems that can provide a simple, rapid, reliable, and efficient method to allow the end-user to generate labeled DNA probes for both CISH and FISH in a short time is also important.

Along with probes, high-quality reagents, fluorescent dyes, buffers, libraries, controls, state-of -the-art automated and digital fluorescence microscopes, and various types of customized options for handling very specific sample types and applications are also being made available for ISH. There are companies that offer ISH services for human and mouse tissue samples.

Chris Silva, Ph.D., vice president, marketing at ACD, mentions the development of new technologies and assays to determine which cells express the target RNA, down to the resolution of a single base. “Next-generation sequencing or qPCR are useful for discovery and quantification of RNA expression and mutations, but they destroy all the important morphological context,” says Silva. “Technologies like BaseScope™ add the critical dimension of morphology to RNA and mutation detection, allowing for assessment of heterogeneity of the sample, even when studying small or difficult targets or for splice variance analysis.”

Previously, most commercial FISH detection kits contained one probe labeled with a single fluorochrome or two probes labeled with two distinct fluorochromes. Now, a FISH method that employs multiple probes, called quantitative multi-gene FISH (qmFISH) is being used to simultaneously detect multiple genes, which is useful for clinical investigations of solid tumors or blood malignancies.⁶

Developments in microscopy and imaging systems and advances in probe labeling efficiency have led to enhancements in ISH, enabling the visualization and detection of genetic changes down to the single-cell level. Single molecule RNA FISH (smRNA-FISH) has been used to measure expression changes in multiple genes within single cells.⁷ Manual evaluation of large numbers of clinical FISH samples can be time-consuming and error-prone, and could lead to variability of data and clinical misdiagnosis. Hence, automated FISH is being developed using microfluidic devices and other approaches, to offer accurate, reproducible, cost-effective and easy-to-obtain results in a clinical setting. Lab-on-chip FISH is 10–20 times more cost-effective than conventional methods, and can be fully integrated and automated for high-throughput and multiplexed detection.⁸ Automation with multiplexing capabilities to perform ISH-ISH or ISH-IHC (immunohistochemistry) is also proving very useful.

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For nearly two decades, researchers have known that ISH, particularly FISH and CGH, have extraordinary potential not only as tools for pure research but also for clinical diagnosis in areas like prenatal diagnosis, cytogenetics, and tumor evaluation. However, the lack of moderately priced systems with high-level accuracy prevented their widespread expansion and use in the diagnostic setting. With improvements in technology and increased affordability of genetic testing, these techniques are set to offer wider applications in the not-too-distant future.

What Are the Various Steps Involved in Performing ISH?

ISH can be performed on a variety of different samples such as, cells, smears, frozen tissue, paraffin-embedded tissue, and more. ISH begins with the preparation of slides and fixation of material to preserve the biological morphology of the sample.

Fixation of Tissue
Several variables must be considered when fixing tissue for ISH and it has to be balanced between accessibility of the probe to the target, integrity of target DNA or RNA, and preservation of morphological details. Freezing the tissue in liquid nitrogen (snap freezing), followed by fixing with 4% paraformaldehyde solution and then cryo-sectioning into thin slices works well and allows the tissue to be used both for ISH and for DNA or RNA extractions.

Probe Selection
Probes are complementary sequences of nucleotide bases that are preselected to bind the target DNA or RNA of interest. When choosing a probe for ISH, the sensitivity, specificity, tissue penetration, reproducibility, stability of hybrids, and the application, must be taken into account. The stability of the hybrid (target-probe) depends on the strength of the bond, which decreases from RNA-RNA to DNA-RNA, and is influenced by hybridization conditions such as concentration of formamide, salt concentration, hybridization temperature, and pH. Probes can be as small as 20–40 base pairs (bp) or up to 1000 bp, although, the optimal size is probably around 50–300 bp. Designing the probe is one of the most critical aspects of the protocol and needs careful design considerations and guidelines to be applied. Probes are readily available for purchase from many different vendors, who also offer custom-designed probes that are purified and ready to use.

Probe Labeling
Once the probe is selected, it has to be labeled with a reporter molecule. There are two main types of labeling- radioisotope labeling and nonisotope labeling. Problems associated with radioisotope labeling include a long exposure time, poor spatial resolution, risk of exposure to radioactivity, and disposal of radioactive waste. For nonisotopic labeling, compounds such as biotin, digoxin and digoxigenin (DIG), alkaline phosphatase and the fluorescent labels, fluorescein (FITC), Texas Red, and rhodamine are used. Nonisotopic labeling is not as sensitive as radioactive labeling, and the hybridization results can be difficult to quantify.

Target and Probe Denaturation
The next step involves denaturation of probe and target DNA, which may lead to loss of morphology. A compromise must be found between hybridization signal and morphology. Alkaline denaturation has traditionally been used. Heat denaturation is simple and effective, but the time and temperature has to be optimized to find the best conditions for denaturation.

Prehybridization
DNA and RNA sequences in the sample are often surrounded by proteins, which mask the target nucleic acid. Therefore, cell-permeabilization procedures are often required. Reagents used to permeabilize tissue are HCl, detergents (Triton or SDS), and Proteinase K. Certain steps can be taken before hybridization to increase hybridization efficiency and reduce nonspecific background staining. A prehybridization incubation is often necessary to prevent background staining, however, it has to be carefully monitored as excess digestion can destroy sample integrity. The prehybridization mixture contains all components of a hybridization mixture except for probe.

Use of Controls
It is very important to use several controls to be sure that the observed labeling of the probe is specific to the target sequence. Specificity controls that use alternate sequence or sense (rather than antisense) probes, or multiple probes for the same gene sequence, are used to ensure that the probe sequence is detecting only the desired target sequence. Sometimes control are used to pretreat sections with nonlabeled probe before treating with labeled probe to eliminate the specific labeling of target sequences, leaving only background labeling. Poly(dT) probes or probes against housekeeping sequences are used as controls to detect sample degradation. Positive controls can also be used to make sure that there is no problem within the ISH technique or protocol.

Hybridization
Hybridization depends on the ability of the probe sequence to anneal with its complementary nucleotide on the sample just below its melting point (T_m). Hybridization is performed by incubating a small amount of solution containing the hybridization probe along with the sample, and it is typically done overnight. The next day, a series of washing steps follow to remove the probe that is not bound to target DNA/RNA. During hybridization and washing, several factors like probe construction, temperature, pH, and buffers all play a crucial role in the specificity of the labeling. The composition of the hybridization solution is critical in controlling the efficiency of the hybridization process.

Post-Hybridization Washing
The labeled probe can hybridize nonspecifically to certain sequences on the sample, and such unstable hybrids or unbound probe can be dissociated and removed by performing washes of various stringencies. Washing should be carried out at or close to the stringency condition at which the hybridization takes place with a final low stringency wash. If washing conditions are too stringent a loss of sensitivity can occur and if they are not stringent enough there can be high background labeling. The stringency of the washes can be manipulated by varying the formamide concentration, salt concentration, and temperature.

Detection and Analysis
Quantitation of the ISH data depends on the type of signal generated by the detection system. For radioactive probes, hybrids are detected auto-radiographically. For nonradioactive probes, the signal is generated directly or indirectly by labeled probes and detected by microscopy. Evaluation of ISH results by bright-field microscopy is preferred for most routine applications because the preparations are permanent. However, fluorescence microscopy is highly sensitive and can be used for multiplexing with spectrally different fluorophores.

It is important to be thorough and cautious when interpreting the results from ISH studies. It’s critical to follow the protocols in minute detail, use the right controls and avoid extrapolation of results. Whenever possible, it is important to cross-validate the findings using complementary techniques and find multiple sources to support the functional interpretation of the ISH data.

References

1. John HA, Birnstiel ML, Jones KW. RNA-DNA hybrids at the cytological level. Nature 1969; 223:582-7. [PMID: 5799530]

2. Gall JG, Pardue ML. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci USA 1969; 63:378-83. [PMID: 4895535]

3. Tanner M, Gancberg D, Di Leo A, Larsimont D, Rouas G, Piccart MJ, Isola J. Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples. Am J Pathol. 2000; 157: 1467-72. [PMID: 11073807]

4. Pinkel D, Straume T, Gray JW. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci U S A. 1986; 83: 2934-8. [PMID: 3458254]

5. Deng W, Shi X, Tjian R, Lionnet T, Singer RH. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells. Proc Natl Acad Sci U S A. 2015; 112: 11870-5. [PMID: 26324940]

6. Hu L, Ru K, Zhang L, Huang Y, Zhu X, Liu H, Zetterberg A, Cheng T1, Miao W. Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine. Biomark Res. 2014; 2: 3. [PMID: 24499728]

7. Cui C, Shu W, Li P. Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications. Front Cell Dev Biol. 2016; 4: 89. [PMID: 27656642]

8. Sieben VJ, Debes-Marun CS, Pilarski LM, Backhouse CJ. An integrated microfluidic chip for chromosome enumeration using fluorescence in situ hybridization. Lab Chip. 2008; 8: 2151-6. [PMID: 19023479]

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