Fluorescence in Situ Hybridization: Cell-based Genetic Diagnostic and Research Applications

Fluorescence in-situ hybridization (FISH) is a molecular diagnostic technique that allows visualization of definite chromosome nucleic acid sequences within a cellular preparation.it involves use of precise annealing of fluorescence labeled DNA probe to complementary targeted sequences. Thus, the genes/sequence of interest could be observed visually using a fluorescence microscope. The most basic components of FISH are targeted DNA sequence & DNA probe. Before the hybridization can happen, the DNA probe has to be labeled by using various means for example: random primed labeling, nick translation or polymerase chain reaction. Two different strategies can be used for labeling: direct method & indirect method.

In indirect labeling, DNA probe is labeled with altered nucleotide that contains a hapten, while indirect labeling nucleotides that have been directly altered to contain a fluorophore are used. These fluorescently labeled probes, i. e., FISH probes are denatured first and subsequently, Combination of the denatured probe & target sequence allows the complementary DNA sequences to anneal. For the probes that have been labeled indirectly, an extra step is required to visualize the non-fluorescent hapten which often uses an enzymatic /immunological detection system. Although FISH is faster with the use of directly labeled probes, indirect labeling has the benefit of signal intensification by using numerous strata of antibodies, and thus, brighter signal production may take place as compared with background levels.

Due to its ability to detect chromosomal aberrations such as gene rearrangement, gene deletion and gene amplification & its high specificity; FISH procedures are widely used in molecular diagnostics to detect and identify:

• Centromeres of a specific chromosome.
• Specific oncogenes (locus-specific probe, useful for (ROS1,ALK), amplifications(HER-2), deletions(CLL) etc.).
• Specific tumor-suppressor genes (locus-specific probe, loss is relevant to tumor progression)
• Whole chromosomes (useful for detection of complex chromosomal rearrangements).

FISH procedures are generally carried out on FFPE (Formalin-fixed paraffin embedded) tissue preparations or cellular preparations. Advantages of FISH analysis in comparison with conventional chromosomal analysis consist of the following:

• Large number of cells can be examined (helpful in detection of residual disease)
• Metaphasic cells are not essential, so aberrations can also be detected in non-dividing cells (beneficial in chronic lymphocytic leukemia)
• FISH can be executed in a quite short amount of time
• Aberrations that are too subtle to be detected by use of conventional cytogenetic analysis, can also be identified

Most of the FISH procedures are carried out in dark to avoid photo-bleaching(probes are highly sensitive to light). The FISH slides are observed under florescence microscope fitted with Excitation and Dichroic filters specific to probe. The FISH Procedure, as performed during internship is as follows:

1. Incubate the FFPE(formalin fixed paraffin embedded tissue) at 700c for 3 hrs. followed by dewaxing in xylene for 10 minutes (twice)
2. Dehydrate the slides in 70%, 85% and 100% for 3 min each followed by air drying
3. Immerse the slides in 0.2M HCL for 20 minutes
4. Immerse the slides in molecular water followed by 2X SSC (sodium saline citrate) for 2 minutes each
5. Immerse the slides in 1M NaSCN for 30 minutes at 800C
6. Immerse the slides in molecular water followed by 2X SSC (sodium saline citrate) for 2 minutes each
7. Immerse the slides in 3.5% pepsin solution at 370C for 10 minutes
8. Immerse the slides in molecular water followed by 2X SSC (sodium saline citrate) for 2 minutes each
9. Immerse the slides in 10% neutral buffered formalin for 10 minutes
10. Immerse the slides in molecular water followed by 2X SSC (sodium saline citrate) for 2 minutes each
11. Dehydrate the slides in 70%, 85% and 100% for 3 min each followed by air drying
12. Add 5µl of appropriate probe onto the slides
13. Incubate the slides in thermobrite© pre-set for hybridization
14. Immerse the slides in wash solutions for 3 minutes each
15. Air dry in dark and add 5µl of DAPI +Antifade
16. Observe the slides under fluorescence microscope.