In Situ Hybridization (ISH): Techniques, Applications & Insights

In situ hybridization: A complete guide

In situ hybridization, especially fluorescence in situ hybridization (FISH) or also chromogen in situ hybridization (CISH) is a special technique in which material of cells or tissues is examined. In situ hybridization can be used to detect changes in DNA or RNA to clarify infectious diseases, tumor or genetic alterations.

What is in situ hybridization?

In situ hybridization is a technique to detect RNA or DNA material of cells or tissues. Therefore, an oligonucleotide with approximately 20 nucleotides in length with the complementary sequence is used. This oligo binds to the DNA or RNA Sequence of interest via base pairing (hybridization). The synthetic or enzymatic produced oligonucleotide (the latter we offer as a kit- DNA FISH Kit) is commonly fluorescent labeled to allow detection of the in situ hybridization. The oligonucleotide is also a called (FISH) probe, which is derived from fluorescence in situ hybridization. Also multiplexing fluorescence in situ hybridization is possible. To assay multiple targets different dyes are attached to the oligonucleotides. This enables in situ hybridization with different probes simultaneously and visualize co-localization within a single cell or specimen. Fluorescence in situ hybridization is an important technique used in genetic testing to diagnose diseases caused by chromosomal differences e.g. Down-syndrome as well as infectious diseases (viral, bacterial, fungal genomes).

 

Types of in situ hybridization techniques

Fluorescence in situ hybridization

Fluorescence in situ hybridization is a method used to visualize specific DNA or RNA sequences directly within cells or tissues. In situ hybridization help to detect chromosomal abnormalities such as deletions, amplifications, and translocations. In situ hybridization is particularly useful for targeting highly repetitive sequences such as telomeres or microsatellites. Properly designed synthetic multi-labeled fluorescence in situ hybridization probes have proven to be superior reagents for rapid FISH analysis. They are also competitive in the fluorescent detection of mRNA in cell systems. For example, baseclick offers multi-labeled oligonucleotides efficiently produced via click chemistry as effective fluorescence in situ hybridization (FISH) probes. Detection of single transcripts in situ using baseclick fluorescence in situ hybridization (FISH) probes is remarkably simple and straightforward. From sample preparation to imaging with wide-field fluorescence microscopy, it takes less than a day to obtain results. An experiment using baseclick in situ hybridization (FISH) probes requires no exotic reagents and can detect single mRNA molecules in four easy steps. In situ hybridization (FISH) probes are oligonucleotides containing two or three reporter molecules, typically a fluorescent dye, which improves signal intensity and increases rRNA accessibility.

Chromogenic in situ hybridization

Chromogenic in situ hybridization (CISH) is very similar to fluorescence in situ hybridization (FISH). Chromogenic in situ hybridization is a method to detect gene sequences in fixed cells and tissues. Instead of a fluorescent label, biotin or digoxigenin are used to produce chromogenic in situ hybridization probes. This means that chromogenic in situ hybridization is performed using indirect labeling, in which antibodies or streptavidin are conjugated. This allows detection using a bright-field microscope instead of fluorescence microscopes used in fluorescence in situ hybridization. Using oligonucleotide probes tagged with biotin, non-specific binding sites must first be blocked using bovine serum albumin. For example, baseclick offers biotin-labeled oligonucleotides efficiently produced via click chemistry as effective chromogenic in situ hybridization probes. For digoxigenin, anti-digoxigenin antibodies with high affinity and specificity are used to detect the chromogenic in situ hybridization probe (CISH). Then, HRP-conjugated streptavidin is used for detection. Both, in situ hybridization probe designs and methods are well known in biological immunoassays, e.g. ELISA.

RNA Scope and advanced methods

Recent advances in RNA-based in situ hybridization have led to the development of techniques like in situ sequencing (ISS). First step in ISS is reverse transcribing mRNA transcripts to cDNA. The ISS method is based on circularized oligonucleotide probes, the so-called padlock probes. These probes have been used to spatially resolve gene transcripts in tissue sections. Padlock probes consist of a complementary target sequence at the 5′ and 3′ end of the probe. If the padlock probe is mixed with target nucleic acids, it forms a circular structure during in situ hybridization. In the prescence of DNA or RNA ligase the previously linear padlock probe is converted into a covalently closed circular oligonucleotide. To retain the benefits of padlock probes without the need for this less performing enzymatic ligation, clickable padlock-style probes are used. Therefore the prope is designed with terminal alkyne and azide moieties at the 5’ and 3’ ends (Figure A). This allows a click reaction and leads to circularization (Figure B). Secondary ClampFISH probe hybridisation is performed to enable fluorescence detection via dye-labelled click-ligated oligos (readout probes) to visualise the RNA target (Figure C). Repeated hybridisation and click rounds increase the fluorescence in situ hybridization intensity. These samples are called clamp FISH probes (click-amplifying fluorescence in situ hybridization).

Also PCR step of the circularized probe allow formation of multiple copies. As usual in classical PCR or also real-time PCR the upstream of a in situ hybridization capture-based detection with microarray is possible. This approach allows for the amplification of specific DNA or RNA sequences before hybridization, enhancing the sensitivity and specificity of the detection process. Next advanced method based on in situ hybridization and ISS is hybridization-based in situ sequencing (HybISS). Also here padlock probes are used. But after ligation, fluorophore conjugated hybridizing bridge-probes (HybISS) bind to the circular padlock probes. This allows fluorescent readout detection of the HybISS probes. This method allows increased flexibility and multiplexing, increased signal-to-noise ratio, all without compromising throughput efficiency of imaging large fields of view.

 

Applications of in situ hybridization

Medical diagnostics

Fluorescence in situ hybridization (FISH) tests are often recommended by doctors depending on the age of the mother. Or if the maternal biochemical serum screening or ultrasound findings were abnormal. The use of fluorescence in situ hybridization tests (FISH) includes, for example, prenatal tests to detect aneuploidy of the chromosomes. Fluorescence in situ hybridization tests are used to diagnose abnormalities of chromosomes 13, 18, 21, but gender determination via the X or Y chromosomes can also be analysed using fluorescence in situ hybridization tests.

Fluorescence in situ hybridization is also useful for diagnosing certain types of cancer and can provide additional information to predict a patient’s outcome and response to chemotherapy drugs. The abnormalities found in cancer cells include translocation. That means that a part of a chromosome has broken off and moved to another chromosome. This abnormality can help medical professionals to identify some types of leukemia, lymphoma and sarcoma. Duplication is also a chromosomal abnormality, e.g. in breast cancer cells. Doctors use fluorescence in situ hybridization tests to choose the best treatment for breast cancer patients. For example, a fluorescence in situ hybridization test can show whether cells have extra copies of the HER2/neu gene, indicating a higher likelihood of response to treatments such as trastuzumab (Herceptin).

Research and genomics

RNA-fluorescence in situ hybridization (RNA-FISH) is an essential and widely used tool for visualizing RNA molecules in intact cells. Detection of endogenous mRNA splice variants has been challenging due to the limits of visualization of RNA- fluorescence in situ hybridization signals and due to the limited number of RNA- fluorescence in situ hybridization probes per target. To screen cellular factors that regulate alternative pre-mRNA splicing of endogenous genes a new fluorescence in situ hybridization method was developed based on high-throughput detection of endogenous splicing isoforms (HiFENS). A high-throughput imaging assay based on hybridization chain reaction (HCR) and used HiFENS probes to screen for cellular factors that regulate alternative splicing of endogenous genes. For accurate detection of splicing outcomes with single cell resolution multiplexing probes are necessary. A prominent model system for the study of alternative splicing is the human FGFR2 (fibroblast growth factor receptor 2) gene.

Personalized medicine

Fluorescence in situ hybridization (FISH) is a method used in the diagnosis of genetic aberrations. Thanks to the fluorescence in situ hybridization method, it is possible to detect tumor-specific anomalies. Of interest are targets of personalized oncology such as gene rearrangements (e.g. ALK, ROS1) reflecting numerous translocation partners, deletions of critical regions (e.g. 1p and 19q), gene fusions (e.g. COL1A1-PDGFB), genomic imbalances (e.g. 6p, 6q, 11q) and amplifications (e.g. HER2). The confirmation of a genetic marker is often a direct indication for the start of a specific, targeted treatment.

DNA Stability and Genetic Integrity

dNTPs are the building blocks of DNA and important for the replication and repair of DNA, especially across cellular divisions and generations. The correct use of dNTPs by DNA ensures the preservation of genetic information and DNA stability.

 

Advantages and challenges of in situ hybridization

Benefits

Fluorescence in situ hybridization (FISH) is a robust method that targets unique sequences of interest which can be employed for visualizing targets, chromosomal and genetic alterations, mitochondrial organelles, infectious diseases of bacteria and pathogenes and tumor. A major advantage of in situ hybridization is that it allows the maximum use of difficult to obtain tissue. Hundreds of different in situ hybridizations can be performed on the same tissue. Tissue libraries can be formed and stored in the freezer for later use. Click chemistry allows probes to be designed with three fluorophores instead of just one. These multichromophore probes can easily be prepared using the Cu(I)-catalyzed azide-alkyne click reaction. These multiple dye probes allow reliable detection and localization of mRNA with only a very small number (5-10) of probe strands. Click tagged fluorescence in situ hybridization is so sensitive that it even enables the in situ detection of viral transcripts just 4 hours after infection. Another advantage of fluorescence in situ hybridization: lower risk of contamination compared to in vitro methods.

Challenges

FISH requires a high-resolution digital camera to capture micrographs of the sample before the fluorescence diminishes and fades. Successful interpretation of FISH experiments is dependent on the quality of the starting materials, hybridization efficiencies, and stringency of post-hybridization washes. Sometimes FISH probes produce a bad signal to noise ratio. It is possible to some extend to increase this ratio by using multiple dye labeled oligo FISH probes, but higher labeling rates can change the chemical behaviour of the oligo and lead to quenching between the dye molecules. FISH analysis is still a time consuming process and assays have to be developed. By learning more about the kintics of in situ hybridization, assays can be optimized by workforce to spend.