An brief introduction to fluorescent in situ hybridization (FISH)

1. Introduction to
Fluorescence in situ hybridization

2. Cytogenetics
 Cytogenetics is a branch of genetics that is
concerned with how the chromosomes relate
to cell behaviour, particularly to their
behaviour during mitosis and meiosis.
 Techniques used include
karyotyping, process of pairing and ordering
all the chromosomes of an organism, thus
providing a genome-wide snapshot of an
individual's chromosomes.
Molecular cytogenetics such as fluorescent in
situ hybridization (FISH) and comparative
genomic hybridization (CGH).

3. Advent of banding techniques
 In the late 1960s, Torbjörn
Caspersson developed a quinacrine
fluorescent staining technique (Q-banding)
revealing unique banding patterns for each
chromosome pair.
 This allowed chromosome pairs of
otherwise equal size to be differentiated by
distinct horizontal banding patterns.
 G-banding (utilizing trypsin and Giemsa/
Wright stain) was concurrently developed
in the early 1970s and allows visualization

4. Beginning of molecular
cytogenetics
 In the 1980s, advances were made
in molecular cytogenetics and there was
beginning of use of fluorescent labeled
probes.
 A probe is a single strand of DNA or RNA
that is complementary to a nucleotide
sequence of interest.
 This change significantly increased the
usage of probing techniques as
fluorescent labeled probes were safer.
 In situ hybridization was invented by

5. Types of ISH
 Today there are two basic ways to
visualize RNA and DNA targets in situ—
 fluorescence (FISH) and
 chromogenic (CISH) detection.
 Characteristics inherent in each method
of detection have made FISH and CISH
useful for very distinct applications. While
both use a labeled, target-specific probe
that is hybridized with the sample, the
instrumentation used to visualize the
samples is different for each method.

6.  Fluorescent in situ hybridization (FISH)
refers to using fluorescently labeled probe
to hybridize to cytogenetic cell
preparations. It is a powerful technique for
localizing specific nucleic acid targets
within fixed tissues and cells, allowing you
to obtain information about gene
expression and genetic loci.

7.  In addition to standard preparations
FISH can also be performed on:
bone marrow smears
enzymatically dissociated tissue
samples
uncultured bone marrow
uncultured amniocytes
cytospin preparations

8. In Situ Hybridization Protocol
 A labeled RNA or DNA probe can be used
to hybridize to a known target mRNA or
DNA sequence within a sample.
 This labeled RNA or DNA probe can then
be detected by using an antibody to
detect the label on the probe. The probes
can therefore be used to detect
expression of a gene of interest and the

10. How To Store a Sample
 Preserving DNA is easy because it is a
highly stable molecule.
 However, preserving RNA is much more
difficult due to presence of RNase enzyme.
This may be found on glassware, in
reagents and on the operator and their
clothing.
 RNase will quickly destroy any RNA in the
cell or the RNA probe itself.
 Therefore, users must ensure they use
sterile techniques, gloves, and solutions.

11. General sample storage when using
frozen sections:
For good results on older slides, the slides
should not be stored dry at room
temperature. They should be stored either
in 100% ethanol at -20°C, or in a plastic
box covered in saran wrap at -20°C or -
80°C. Slides stored in this way can be used
for several years.

12. Choice of probe
 RNA probes: RNA probes should be
between 250 to 1500 bases in length.
Probes approximately 800 bases long
exhibit the highest sensitivity and specificity.
 DNA probes: DNA probes can also provide
high sensitivity for in situ hybridization..
However, they do not hybridize as strongly
to the target mRNA molecules as RNA

14. Fiber FISH
 Interphase chromosomes are attached to a
slide in such a way that they are stretched out
in a straight line, rather than being tightly
coiled, as in conventional FISH
 A technique known as chromosome
combing is used for this purpose.
 The extended conformation of the
chromosomes allows dramatically higher

15. MA-FISH
 Microfluidics-assisted FISH (MA-FISH)
uses a microfluidic flow to increase DNA
hybridization efficiency, decreasing
expensive FISH probe consumption and
reduce the hybridization time.
 MA-FISH is applied for detecting
the HER2 gene in breast cancer tissues.

16. Hybrid Fusion-FISH
 Hybrid Fusion FISH (HF-FISH) uses primary
additive excitation/emission combination of
fluorophores to generate additional spectra
through a labeling process known as dynamic
optical transmission (DOT).
 It enables highly multiplexed FISH applications
that are targeted within clinical oncology panels.
 The technology offers faster scoring with efficient
probesets that can be readily detected with

17. Medical applications of FISH
FISH can be used to form a diagnosis, to
evaluate prognosis, or to evaluate remission of a
disease
Examples of diseases that are diagnosed using
FISH include
 Prader-Willi syndrome
 Angelman syndrome
 22q13 deletion syndrome
 Chronic myelogenous leukemia
 Acute lymphoblastic leukemia
 Cri-du-chat
 Velocardiofacial syndrome

19. Comparative genomic
hybridization
 Uses FISH in a parallel manner with the
comparison of the hybridization strength
 To recall any major disruptions in the
duplication process of the DNA
sequences in the genome of the
nucleus.

20. Spectral karyotype
 Spectral karyotyping is an image of
colored chromosomes.
 It involves FISH using multiple forms of
many types of probes with the result to
see each chromosome labeled through its
metaphase stage.
 This type of karyotyping is used