I. What are the main factors that affect FISH?

FISH is affected by light exposure, temperature, humidity, and the pH of various reagents. Temperature and humidity directly affect the hybridization efficiency between the probe and the target DNA; light exposure affects the intensity of the fluorescent dye, so probes must be stored away from light, and hybridized slides should be sealed with a fluorescence quencher and stored away from light; The pH of all reagents must also be precisely maintained within the required range, as this directly affects the stability of the FISH assay.

II. What is the basic principle of fluorescence in situ hybridization?

FISH is a commonly used technique that employs fluorescently labeled DNA probes to detect genetic variations that are typically beyond the resolution of conventional chromosome banding analysis. Its principle is based on the recognition and binding of single-stranded DNA molecules to complementary sequences on metaphase chromosomes or in interphase nuclei.

The probe and target DNA are treated with a hot formamide solution to denature the double-stranded DNA. The probe and target DNA are then incubated at 37°C to anneal through complementary base pairing between the probe and the target sequence. A fluorescence microscope equipped with appropriate filters is used to detect hybridized probes that produce a visible signal on the target. A single target can be probed simultaneously with multiple probes labeled with different fluorescent colors to detect one or more specific regions of the genome.

FISH is frequently performed on chromosomes in the metaphase of cultured cells, allowing the probe signal to be localized directly to the chromosome to detect congenital or acquired chromosomal alterations. FISH can also be applied to target genomic sequences in non-dividing cells to identify chromosomal abnormalities independent of the cell cycle phase. This technique, known as interphase FISH (iFISH), is used for chromosome counting and the identification of chromosomal rearrangements in various clinical specimens.

III. What is the difference between DNA-FISH and RNA-FISH?

Both DNA-FISH and RNA-FISH can visually display multiple targets in the same sample and are reusable; the former can be observed using fluorescence microscopy, while the latter can be observed using fluorescence microscopy as well as for high-content screening (HCS) and flow cytometry analysis. DNA-FISH is primarily used for gene presence, copy number, and location identification, as well as mutation analysis; RNA-FISH is primarily used to detect gene expression and the temporal and spatial localization of RNA.

IV. What are the main classifications and uses of DNA-FISH probes?

Centromere probes (CSP probes): Used to detect chromosomal number abnormalities such as trisomy and monosomy;

Chromosomal arm or whole-chromosome staining probes (WPP probes): Used to detect chromosomal translocations and label chromosomes;

Sequence-specific probes (GLP probes): These include various types such as arm, band, and gene probes;

In addition, there are subtelomeric probes; the 200–300 Kb region near the telomere consists of chromosome-specific DNA and is used to detect cryptic translocations involving telomeres.

V. What is the difference between directly labeled probes and indirectly labeled probes?

Directly labeled probes refer to DNA probes covalently linked to a fluorophore; indirectly labeled probes refer to DNA probes first linked to a hapten, such as digoxin or biotin, and then the hapten is linked to a fluorophore, thereby forming a probe-hapten-fluorophore complex resembling a “sandwich.”

Compared to indirect-labeled probes, direct-labeled probes are characterized by low background and high specificity. With the continuous advancement of fluorescent dyes and fluorescence detection technologies, their sensitivity has steadily improved. Consequently, in the field of FISH testing—particularly in disease genotyping—probes are predominantly designed using the direct-labeling approach.

VI. What is the procedure for performing FISH on peripheral blood samples?

1. Carefully remove the coverslip from the hybridization area using forceps;

2. Immerse the slide in 70%, 85%, and 100% ethanol for one minute each to ensure thorough dehydration. Allow the slide to air-dry before proceeding with repeated hybridization. For secondary hybridization, it is recommended to reduce the denaturation time by half, but it must not be less than 3 minutes. If a third hybridization is to be performed on the same sample, there is no need to reduce the denaturation time. For multiple hybridizations, reducing the concentration of the counterstain solution helps maintain probe brightness. FISH can also be performed on slides that have already undergone G-banding: Immerse the slides in 70%, 85%, and 100% ethanol for two minutes each to ensure thorough dehydration. Allow the slides to air-dry before proceeding with repeated hybridizations. For the second hybridization, it is recommended to reduce the denaturation time by half, but it must not be less than 3 minutes. If a third hybridization is to be performed on the same sample, there is no need to reduce the denaturation time.

VII. What could be the cause of a rapid decline in the fluorescent signal over a short period of time?

Under normal circumstances, if the probes are stored properly after hybridization, the fluorescent signal can be maintained for more than six months. A rapid decline in the signal is primarily due to a failure to strictly avoid light exposure during the procedure, observation, or storage of the probes.

Sunlight or strong artificial light can cause rapid quenching of the fluorescent dye, leading to unreliable observation results. Therefore, operations and observations should be conducted in a darkroom whenever possible. Alternatively, an anti-quenching agent can be added during mounting to delay the quenching of the fluorophore.

VIII. What are the advantages of FISH technology?

(1) Safe, rapid, and highly sensitive;

(2) Probes can be stored for extended periods;

(3) Multicolor labeling, simple and intuitive;

(4) Can be used for the analysis of metaphase chromosomes and interphase cells;

(5) Applicable to a wide range of materials, including fresh, frozen, or paraffin-embedded specimens, as well as fine-needle aspiration samples and exfoliated cells.

KMD Bioscience has been dedicated to research in cell biology technologies for many years. With a team of experienced scientific experts, a highly skilled laboratory team, a comprehensive cell culture platform, and state-of-the-art equipment and experimental conditions, we have accumulated extensive experience in fluorescence in situ hybridization (FISH) technical services and established a comprehensive FISH technical service platform. We are able to provide one-stop FISH technical services ranging from probe design, sample processing, hybridization detection, gene expression studies, and data analysis. By standardizing and streamlining the testing procedures, we have enhanced the accuracy of experimental results, reduced false positives and false negatives, and ensured that we provide our clients with high-quality FISH services. Our fluorescent reagents and probes are cost-effective and safe, allowing experiments to be completed in a short time. This helps clients reduce the complexity and time required for in-house FISH procedures, while also lowering experimental costs, conserving valuable samples, and supporting their research efforts.

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