To preserve morphology, fresh tissue should be rapidly removed and fixed as soon as possible. For paraffin-embedded tissue sections, formalin fixation is always used. There is still no fixation protocol which can be used for all substrates, and the fixation protocols must be optimized for different applications. After fixation, the sample is embedded in paraffin or OCT for long-term storage and sectioning for subsequent procedure.
It is known that the target DNA or RNA sequences are surrounded by proteins and the extensive cross-linking of these proteins mask the target nucleic acid, which present obstacles to good infiltration of the probe. Therefore, permeabilization procedures are critical to in situ hybridization. Three main reagents used to permeabilize tissue are proteinase, HCl and detergents. Protease treatment serves to increase target accessibility by digesting the proteins that surround the target nucleic acid.
Proteinase K or pronase is usually used to remove those proteins. Incubation has to be carefully monitored because if the digestion proceeds to far you could end up destroying most of the tissue or cell integrity. In some protocols a min treatment with 0. Although the precise action of the acid is unknown, the extraction of proteins and hydrolysis of the target sequence may contribute to a decrease of the level of background staining. The Triton X, SDS and other detergents, are frequently used to permeabilize the membranes by extracting the lipids membrane.
This is not usually required in tissue that has been embedded in wax, but it is critical to intact cells or cryostat sections. Probe specificity is important. If the exact nucleotide sequence of the mRNA or DNA in the cell is known, a precise complementary probe can be designed. This means the probe is more likely to be washed away during wash and detection steps and may not be correctly detected. This protocol describes the use of DIG-labeled single-stranded RNA probes to detect expression of the gene of interest in paraffin-embedded sections.
For frozen sections, start at Step 2. If using formaldehyde-fixed paraffin-embedded sections, continue with this step. Before proceeding, slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section. Keep the slides in the tap water until antigen retrieval. From this point onwards the slides cannot dry as this will cause non-specific antibody binding and high background staining.
Incubation time and proteinase K concentration may require optimization. We recommend trying a proteinase K titration experiment to determine optimal conditions. Insufficient digestion will reduce hybridization signal and over-digestion will result in poor tissue morphology, making localization of the hybridization signal very difficult. Cancer is considered a genetic disease at the cellular level resulting from either a progressive process or a one-off catastrophic event Stephens et al. The two main pathogenetic pathways for hallmarks of cancer development are the inactivation of tumor suppressor genes by deletions, mutations, miRNA upregulation, or epigenetic mechanisms, and the activation or deregulation of oncogenes as a consequence of point mutations, amplification or balanced cytogenetic abnormalities Vogelstein and Kinzler, ; Hanahan and Weinberg, Recurrent chromosomal abnormalities including translocations, deletions, duplications, and gene amplifications associated with distinct tumor entities have been characterized; specifically designed FISH panels have been widely used in the diagnosis and monitoring of acquired chromosomal abnormalities in hematologic and solid tumors Hu et al.
Current guidelines recommend an integrated approach for cancer cytogenetic diagnosis Wolff et al. In general, both conventional karyotyping and FISH testing are used for initial diagnosis and follow up monitoring of clonal abnormalities. For hematopoietic and lymphoid tumors, the most commonly used FISH probes and disease-specific panels in a clinical cytogenetics laboratory are listed in Table 1.
Results from a FISH panel offer a quick evaluation of targeted abnormal patterns and their percentage within the bone marrow cells or leukocytes. Chromosome analysis will then reveal the clonal abnormalities and clonal evolution. Adjunctive use of FISH probes to further define ambiguous or hidden chromosomal abnormalities is required for many cases Kamath et al.
Additionally, FISH is a sensitive and timely method to monitor residual diseases with known clonal abnormality and bone marrow transplantation by sex-mismatch donor at cellular level. For example, cyclin D1 CCND1 translocation can be detected by FISH as a characteristic abnormality in mantle cell lymphoma, which provides differential diagnosis for morphologically similar chronic lymphoid leukemia CLL. Furthermore, FISH for nuclear DNA can be combined with immunostaining of cytoplasmic markers for simultaneous identification of chromosomal abnormalities and cell types.
This modified immuno-FISH was expected to improve the diagnostic accuracy but the low sensitivity limited its application only in follow-up study Boersma-Vreugdenhil et al.
FISH tests are widely used in various types of solid tumors. For example, FISH can define gene rearrangements in congenital fibrosarcoma with a novel complex translocation Marino-Enriquez et al.
FISH results can be used to guide cancer treatment. For example, Herceptin-targeted therapy is effectively against HER2 over-expressed breast cancer. Many targeted therapies for recurrent translocations in various types of solid tumors have been either approved by FDA or are under clinical trials.
For example, lapatinib, sorafenib, sunitinib, termsirolimus, and pazopanib have been used for papillary renal cell carcinoma with translocations involving the TFE3 gene at Xp FISH assays using probes for specific recurrent translocations from different solid tumors could guide effective targeted therapy.
FISH tests were also used to evaluate sperm aneuploidy frequencies before and after chemotherapy in patients with testicular cancer and Hodgkin's lymphoma; significantly increased frequencies of aneuploidies for a duration up to 24 months were noted De Mas et al.
It was recommended that genetic counseling about potentially increased reproduction risk from chemotherapy should be offered to cancer patients. The majority of FISH probes target to specific chromosomal and genomic abnormalities in the human genome. Rapid phylogenetic identification of single microbial cells was achieved using fluorescently labeled oligonucleotides complementary to 16S ribosomal RNA rRNA DeLong et al. Some segments in the 16S rRNA are invariant in all organisms but phylogenetic group-specific 16S rRNA in different groups of organism can be used as oligonucleotide FISH probes length 17—34 nucleotides to identify infectious agents in clinical samples.
FISH assays using locus-specific and regional painting probes are still a powerful tool in visualizing simple and complex chromosomal and genomic rearrangements. Fiber-FISH by locus-specific BAC clone probes within a Kb 17q12 inversion hybridizing onto stretched DNA fibers correlated the inversion orientations with associated haplotypes, which allowed the evaluation of inversion frequencies among human populations globally Donnelly et al.
Pericentriomeric heterochromatin probes were used in a three dimensional FISH 3D-FISH to study intra-nuclear centromeric positions in cultured cells from patients with ICF syndrome immunodeficiency, centromeric region instability, facial anomalies and Robert syndrome cohesion defect by mutations in the ESCO2 gene Dupont et al. Chromothripsis are seen as regional clustering of breakpoints and regularity of oscillating copy-number states by microarray analysis and as heterogeneous staining regions, marker or ring chromosomes, and other undefinable rearrangements by chromosome analysis Stephens et al.
Selected FISH probes targeting to the oscillating copy-number gains and losses could be used to monitor the abnormal clones with chromothripsis. FISH technology has made significant progress with the innovation of novel labeling methods and the introduction of super resolution imaging systems for fine mapping of intra-nuclear genomic structures and for single cells single molecule profiling of cytoplasmic RNA transcription. This method enabled the visualization of intra-nuclear locations and dynamics of telomeres and MUC4 loci during mitosis in living human cells Chen et al.
However, using tiling sgRNAs for single-copy gene regions could have low labeling efficiency and higher background. Figure 2. Single molecule FISH techniques for research application in single cells. B Oligopaint-FISH using fluorophore-coupled primary oligonucleotides for targeted SNP loci and fluorophore-coupled second oligonucleotide to enhance labeling efficiency shows differential labeling of paternal pat and maternal mat chromosomes.
C single molecule RNA FISH by rolling cycling amplification RCA using padlock probes targeting to reverse transcripted cDNA with different alleles followed by ligation, cycling amplification and specific fluorophore-couple probe hybridization and visualization.
D Sequential barcoding of multiplex different mRNAs by repeat rounds of hybridization, imaging, and stripping. Star, diamond, and triangle are symbols for different fluorophores. A synthesized primary single-strand oligonucleotide library targeting to a single copy region of the genome along with fluorophore-coupled second oligonucleotides complementary to a portion of the primary oligonucleotides were developed for so-called oligopaint FISH Beliveau et al.
Oligopaint FISH probes designed with one fluorophore for specified single nucleotide polymorphisms SNPs in a targeted region from one chromosome and another fluorophore for these SNPs in the homology chromosome enabled differential labeling of the two homologous chromosomes.
Stochastic optical reconstruction microscope STORM was used for single-molecule super-resolution imaging. Therefore, with prior information of the specific SNP alleles from the two homologous chromosomes, oligopaint FISH showed in situ haplotyping for paternal and maternal chromosomes Figure 2B. The oligopaint probes are chosen bioinformatically to avoid repetitive DNA sequences and they can be selected to target any organisms whose genomes have been sequenced.
With further improvement on signal pattern recognition from the SNP loci, oligopaint FISH should enable direct analysis of fine-scale chromatin structure, differential visualization of homologous chromosomes, and allele-specific studies of gene expression. Several approaches, including branched DNA probes, tyramide signal amplification, quantum dots, and padlock-rolling circle amplification RCA , have been used for signal enhancement Kwon, RCA is the only method capable of distinguishing single nucleotide allelic changes in transcripts.
To increase the capacity for multiplex detection of different mRNA molecules in single cells, combinatorial labeling, and optical super-resolution microscope were used to measure mRNA levels of 32 genes simultaneously in single Saccharomyces cerevisiae cells Lubeck and Cai, Further modification introduced a sequential barcoding scheme for multiplex different mRNA quantitation Lubeck et al. In this scheme, the mRNAs in cells were barcoded by sequential rounds of hybridization, imaging and probe stripping Figure 2D.
Theoretically, the multiplexing capacity scaled up quickly as the number of fluorophores and rounds of hybridization increased. In practice, the available fluorophores were limited and each round of hybridization introduced loss of the RNA integrity in the tested cells.
Additionally, smRNA FISH has been used to study the subcellular localization and cell-to-cell variability of long non-coding RNAs lncRNA ; systematically quantification and categorization based on the subcellular localization patterns were achieved for a representative set of 61 lncRNAs in three different cell types Cabili et al. Knowledge of lncRNA subcellular localization patterns is essential to understand its biological processes.
Real-time live imaging using laser-scanning confocal microscope with photon-counting detectors for quantitative studies of transcription in culture cells and model animals have been achieved by smRNA-FISH and GFP-tagged reporter gene for RNA polymerase Gregor et al. Using Drosophila embryo as a testing system, smRNA-FISH observed stochastic transcriptional activity of four critical patterning genes and co-packaging of transcripts as multi-copy heterogeneous granules to selected subcellular domains Little et al.
These results indicated that there are conserved mechanisms of precision mRNA transcription and localization for spatiotemporal control of protein synthesis in regulating cellular and embryo development. FISH has the advantage that it can be used in metaphase chromosomes and interphase nuclei, and thus offers a cell-based genetic diagnosis in complementary to DNA-based molecular testing Xu and Li, FISH has been used as adjunctive and diagnostic assays for both constitutional and somatic cytogenomic abnormalities.
FISH analysis of uncultured interphase cells from amniotic fluid or chorionic villus samples is a standard procedure for rapid prenatal testing of common aneuploidy and genomic disorders, which alleviates much anxiety for patients and physicians.
The use of interphase FISH has been particularly fruitful for cancer cytogenetics, where the detection of recurrent chromosomal abnormalities and clonal evolution is crucial for classifying different types of tumors, selecting treatment protocols, and monitoring outcomes. Even with the introduction of genomic technologies like microarray analysis and exome sequencing, FISH analysis will still be an integral part of genetic diagnosis Parisi et al.
Microfluidic devices for miniaturized and automatic FISH applications are currently under development Vedarethinam et al. The validation of these devices in the near future and the available of more disease-specific probes will further enhance and expand the diagnostic FISH application.
Novel FISH techniques and super-resolution imaging systems have been introduced to study the spatiotemporal changes of intra-nuclear genomic organization and cytoplasmic RNA profiling. The translation of these single molecule single cells FISH techniques into cell-based genetic diagnosis is expected to improve the analytical resolution and capacity for a spectrum of genetic defects from chromosomal and genomic abnormalities to epigenetic aberrations. PL organized, modified, and edited the manuscript.
We would like to thank Audrey Meusel for proofreading and editing this manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Nature Education 1 1 Cytogeneticists can now go "FISH-ing" for chromosomal abnormalities, which are deletions and duplications that can cause disease. How exactly does FISH work? Aa Aa Aa. Fluorescent Probes Are Introduced.
Two labeling strategies are commonly used: indirect labeling left panel and direct labeling right panel. For indirect labeling, probes are labeled with modified nucleotides that contain a hapten, whereas direct labeling uses nucleotides that have been directly modified to contain a fluorophore.
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