In situ hybridization (ISH) uses labeled DNA or RNA to probe a biological sample for the presence of a complementary sequence. The technique was first developed almost 50 years ago and employed radioactive labels for detection, however these were rapidly superseded by fluorescent labels to afford greater safety, stability, and ease of detection. Fluorescent in situ hybridization (FISH) has recently seen a surge in popularity due to advances in oligo synthesis technology and improvements in bioinformatics algorithms. While currently it is most commonly performed using fluorescently labeled DNA probes, its capabilities continue to expand as interest in RNA FISH and transcriptome analysis grows.
According to Jessica Kaplunov, product marketing manager at LGC Biosearch Technologies, FISH analysis within a research setting would most commonly be performed on cells grown in tissue culture or on fresh-frozen tissue samples, yet customers are continually discovering new applications for the technique. “Whole-mount samples, usually naturally clear tissues such as larvae, eggs, and embryos, are becoming increasingly common among users of LGC Biosearch Technologies’ Stellaris® RNA FISH products, while CLARITY-cleared mouse brains, 3D cellular models, plant tissues, and viruses have also been analyzed,” Kaplunov explains. “We work closely with researchers to co-develop new applications and capabilities of RNA FISH, 
and there is considerable excitement and enthusiasm among thought leaders and within our group for approaches such as CASFISH, seqFISH, and MERFISH, all of which hold possibilities for advancing the technology.”
Image: HER2 mRNA (green) and HER2 protein (magenta) using Stellaris RNA FISH and immunofluorescence in human cells.
A typical FISH assay employs a relatively straightforward protocol utilizing fluorescent probes to bind chromosomes only in regions of high sequence complementarity. In this methodology, cells are fixed and permeabilized on a slide, washed, and then placed in pre-hybridization buffer (a saline sodium citrate solution that contains formamide). This is followed by hybridization of the probe with sample DNA, wherein both the probe and the target DNA are first denatured in the presence of hybridization buffer, allowed to hybridize, and then treated to a series of washes. Mounting media is then added to the slide and fluorescence is visualized using an epifluorescence microscope equipped with filters appropriate to visualization of the probe-dye conjugates. Fluorescent readouts are highly amenable to multiplexing yet issues relating to spectral overlap can impose limitations on the output of a FISH assay. Overcoming this constraint is the primary aim of the innovative seqFISH and MERFISH approaches mentioned above, both of which use sophisticated fluorescent barcoding methodologies to increase the throughput of gene expression profiling.
“One major strength of RNA FISH is that it addresses the question of stochastic expression in a way that more classic techniques are unable to do,” says Kaplunov. “While we’ve known for a long time that expression of proteins and RNA between cells and even within a single cell can vary tremendously, techniques have routinely involved combining millions of cells together into one data point. Stellaris RNA FISH allows researchers to visualize expression patterns and target localization, and to quantify individual RNA molecules in situ to gain a much more nuanced understanding of expression and sequestering. Results generated with FISH add significant value to research, and this is exemplified by the exponential growth which we’ve seen in the number of papers citing Stellaris RNA FISH year on year.”
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Stellaris RNA FISH is quantifiable, capable of high order multiplexing, and offers exceptional specificity through a patented 48 probe technology. “This tiles up to 48 unique probes along the target RNA, each of which has a single small fluorophore, and only when dozens of probes are bound does the combined fluorescence become visible as a spot,” elaborates Kaplunov. “A Stellaris RNA FISH assay can be completed in a single day; furthermore, it can be combined with immunofluorescence to generate images of both RNA and proteins labeled together.”
Genetic testing and more
“In a clinical setting, FISH is used for genetic testing to determine duplications, deletions, and translocations on chromosomal spreads from patient cells derived from biopsies, blood, or urine specimens,” notes Nadia Rana, product manager at Enzo Life Sciences. “This is applicable to prenatal testing, oncology screening, or even developmental disability diagnoses. FISH represents a rapid screening technique that is cost-effective compared to reflexive testing, including next-generation sequencing. It has particular utility when a clinician or researcher has a specific gene of interest for monitoring aneuploidy, as opposed to examining the genome for aberrations.”
Enzo has an extensive portfolio of FISH products that includes probes designed for hematology, solid tumor, as well as prenatal, postnatal, and preimplantation genetic testing. In addition, the company’s Nick Translation DNA Labeling System 2.0 can be used for the synthesis of custom probes against novel targets, in under an hour. The latter can accommodate a wide range of fluorophore-labeled, biotin-labeled, and digoxigenin-labeled nucleotides, providing significant flexibility. Enzo is also poised to launch a new quadruple color probe for the detection of changes in copy number of CDKN2A, a gene that is frequently found to be mutated or deleted in bladder cancer cells. “Our DEEPSEE™ CDKN2A/CEN3/7/17 Quad Probe incorporates our proprietary SEEBRIGHT™ dyes to give clear, bright signals for detecting copy numbers of the tumor suppressor gene CDKN2A at the 9p21 locus of chromosome 9, and enumeration probes CEN3, CEN7, and CEN17 for detection of aneuploidy of chromosomes 3, 7, and 17,” says Rana.
Probes and libraries
Arbor Biosciences is another company that offers significant expertise in FISH. Its flexible myTags® product range includes ready-to-use labeled FISH probes in addition to immortal libraries that can be labeled in-house. “We use up-to-date bioinformatics algorithms for probe design to ensure high specificity to the sequence of interest,” explains Kassandra Semrau, product manager, “and all of our libraries go through rigorous quality control including NGS to verify their quality.”
Semrau points out that FISH offers several significant advantages over other laboratory techniques. “Genome assemblies rely on sequence read overlap, but it can be difficult to piece scaffolds into linkage groups if you lack sufficient read depth or consensus sequence,” she says. “By designing highly specific FISH probes you can use multicolor FISH for genetic mapping or genome assembly of scaffolds, while RNA FISH can provide information about gene expression events that aren’t discernible from RNA sequencing. The only limitation to FISH is that you need to generate a unique spectral signal for each target, which techniques such as MERFISH are overcoming with barcoding fluorophores.”
According to Semrau, the assessment of transcriptome networks is likely to be the primary driver to advancements in FISH technology, with CASFISH, seqFISH, and MERFISH already providing examples of improved methods of visualizing DNA and RNA sequences. Kaplunov adds that she particularly likes the use of seqFISH to identify targets of interest followed by Stellaris RNA FISH assays to visualize, localize, and quantify those targets in situ. “Because Stellaris is the only amplification-free RNA FISH technique, it is a true orthogonal validation to RNA-seq. qPCR, which is typically used as a validation tool, also uses primer-based amplification and could represent the same biases as RNA-seq itself. Stellaris bridges the gap between more classic techniques and the stochastic expression and cell-specific data gained by FISH, and represents a logical next step in laboratory technique, providing a full range of data.”
Image (top) courtesy of Daniel Mietchen, [CC BY-SA 2.5], via Wikimedia Commons.