In situ Hybridization (ISH)
description
Transcript of In situ Hybridization (ISH)
ond sI n itu Hybri i izat
Dr.Meera N riaResearch Associate, SCTL., C.S.R.D. People’s Group, Bhopal
In situ Hybridization
In situ Hybridization
In its original place; i.e. in position
+
The process of forming a double stranded nucleic acid from joining two
complementary strands
of DNA (or RNA).
HybridizationHybridization
Normal hybridization requires the isolation of DNA or
RNA, separating it on a gel, blotting it onto
nitrocellulose and probing it with a complementary
sequence
In solution - In vitro
On cell preparations or tissue sections -
In situ
On nitrocellulose membranes
Southern Blotting: DNA
Northern Blotting: RNA
Western Blotting: Proteins
HybridizationHybridization
Nucleic Acid HybridizationNucleic Acid Hybridization
Two DNA or RNA single chains from different biological
sources, make the double catenary configuration, based on
nucleotide complementarity and of contingent sequence
homology of the two sources
Principle:Principle:
To identify a particular recombinant clone if a DNA or RNA
probe, complementary to the desired gene, is available.
Two basic notions are used:
Target molecule
DNA, RNA or protein sequence
that is to be identified
Purpose : Identification or localization of certain nucleic
acid sequences in the genome of some species.
Probe
Identify the target, by hybridization.
Target molecule ProbeTarget molecule Probe
TypesTypes
DNA:DNA hybrids
DNA:RNA hybrids
RNA:RNA hybrids
At High Temperatures
At cooling
This is the basis for DNA probe hybridization
Non-homologous DNA do not attach
This technique help in determining the gene structure and
in identifying molecules which contain same sequence of
nucleotide.
Annealing is the physical process responsible for the
association of two complementary sequences
The two complementary sequences form hydrogen bonds
between their complementary bases (G to C, A to T or U).
The hydrogen bonding form a stable double-stranded, anti-
parallel "hybrid" helical molecule.
CreditsCredits Buongiorno-Nardelli and Amaldi – Nature
Gall and Pardue – PNAS
John, Birnstiel and Jones - Nature
In situ hybridization (ISH) is a type of hybridization that uses
a labeled complementary DNA or RNA strand (i.e., probe) to
localize a specific DNA or RNA sequence in a portion or
section of tissue (in situ).
Section : - a small tissue
- or entire tissue (whole mount ISH),
- in cells and
- in circulating tumor cells (CTCs).
This is distinct from immunohistochemistry
In order to probe the tissue or cells of interest one has to
increase the permeability of the cell and the visibility of the
nucleotide sequence to the probe without destroying the
structural integrity of the cell or tissue.
Probe to use, and how best to label it, to give the best level of
resolution with the highest level of stringency.
Things to keep in mind Technique is sensitive : Threshold levels of detection are in
the region of 10-20 copies of mRNA
per cell.
Stages in ISHStages in ISH Preparation of MaterialPreparation of Material Choice of Probes Labeling Fixation Hybridization Detection
Common tissue sections used are:
Preparation of Material
a) Frozen sections
b) Paraffin embedded sections
c) Cells in suspension
a) Frozen Sections:
Preparation of Material
Fresh tissue is snap frozen (rapidly put into a -
80 freezer)
Embedded in a special support medium for
thin cryosectioning.
Sections are lightly and rapidly fixed in 4%
paraformaldehyde just prior to processing for
hybridization.
b) Paraffin embedded sections :
Preparation of Material
Sections are fixed in formalin.
Embedded in wax (paraffin sections) before
sectioning.
c) Cells in suspension :
Cells can be cytospun onto glass slides.
Fixed with methanol.
Preparation of Material Choice of ProbesChoice of Probes Labeling Fixation Hybridization Detection
Stages in ISHStages in ISH
These are short synthetic, single stranded oligonucleotides
which are labelled.
These probes can be as small as 20-40 base pairs or be up
to 1000 bp.
Strength decreases in the order RNA-RNA to DNA-RNA.
Stability depends on various hybridization conditions
Choice of Probes
How it works
Probe is used to identify a complementary sequence
i.e target DNA
Optimization of the conditions is must.
Strength of the bonds between the probe and the target
plays an important role.
In situ hybridization probes
I. Double-stranded DNA (dsDNA) probes
II. Single-stranded DNA (ssDNA) probes
III. Synthetic oligonucleotides
IV. RNA probes (Riboprobes)
DNA ISH
RNA ISH
I. Double-stranded DNA (dsDNA) probes
Double stranded
Requires denaturation prior hybridization
Probes are less sensitive
Not widely used today.
Produced:
If sequence is known then by designing appropriate
primers one can produce the relevant sequence very
rapidly by PCR, potentially obtaining a very clean sample.
By the inclusion of the sequence of interest in a bacteria
Sequence of interest is excised with restriction enzymes.
Replication Cells are lysed DNA extracted, purified
Easy to use
Sub-cloning unnecessary
Choice of labeling methods
High specific activity
Possibility of signal amplification (networking)
Production of large quantities of probe sequence in
question.
Advantages of ds DNA probes
Disadvantages ds DNA probes Probe denaturation required
Re-annealing during hybridization
Increasing probe length and decreasing tissue
penetration
Less sensitive
Hybrids less stable than RNA probes
Much larger, 200-500 bp size range
Produced by :
Reverse transcription of RNA or
Amplified primer extension of a PCR-generated
fragment in the presence of a single antisense primer.
Preparation time is long.
II. Single-stranded DNA (ssDNA) probes
No probe denaturation needed
No reannealing required during hybridization (single
strand)
Advantages of ss DNA probes
Disadvantages ssDNA probes
Technically complex
Subcloning required
Hybrids less stable than RNA probes
Expensive.
III. RNA probes These are ss RNA probes prepared by transcription from
cloned DNA, which complements a specific mRNA or DNA
Also called as complementary RNA (cRNA) or Riboprobes
(Promega Corporation).
It is used for studies of virus genes, distribution of specific
RNA in tissues and cells, integration of viral DNA
into genomes, transcription, etc.
Two methods for preparing RNA probes:
Complimentary RNA's are prepared by an RNA polymerase-
catalyzed transcription of mRNA in 3' to 5' direction.
In vitro transcription of linearized plasmid DNA with RNA
polymerase can be used to produce the RNA probes. Here
plasmid vectors containing polymerase from bacteriophages
T3, T7 or SP6 are used
Advantages of RNA probes
Stable hybrids (RNA-RNA)
High specific activity
No probe denaturation needed
No reannealing
Un-hybridized probe are enzymatically destroyed, sparing
hybrid.
Most widely used probes
Disadvantages RNA probes
Very difficult to work.
Subcloning needed.
Less tissue penetration.
IV. Oligonucleotide Probes These are short sequence of nucleotides that are synthesized to
match a specific region of DNA or RNA then used as a
molecular probe to detect the specific DNA or RNA sequence.
These are produced synthetically by an automated chemical
synthesis.
Small generally around 40-50 base-pairs.
Ideal for in situ hybridization because of their small size which
allows easy penetration into the cells or tissue of interest.
Advantages of Oligonucleotide Probes
No cloning or molecular biology expertise required.
Stable
Resistant to RNases
Good tissue penetration (small size)
Constructed according to recipe from amino acid data
No self-hybridization
No renaturation
Readily available, faster and less expensive to use.
Easier to work with
More specific
Better tissue penetration,
Better reproducibility
Wide range of labeling methods that do not interfere with
target detection.
Disadvantages of Oligonucleotide Probes
Limited labeling methods
Lower specific activity, so less sensitive
Dependent on published sequences
Less stable hybrids
Access to DNA synthesizer needed
Probe generation In vitro Transcription
Probe synthesis using Randomized primers
Synthesizer
PCR
Stages in ISHStages in ISH Preparation of Material Choice of Probes Labeling Labeling Fixation Hybridization Detection
Labeling A specific probe is used to locate the presence, and in
some cases, quantify the amount of target on the blot.
The choice of the label depends on many factors such as
sensitivity, quantification requirements, ease of use, and
experimental time.
The choice of the label depends on many factors such as
sensitivity, quantification requirements, ease of use, and
experimental time.
Labeling of Probes End Labeling: used to label oligonucleotide probes
5' end labeling
3' end labeling
3' end labeling
Photobiotin Labeling: Chemical reaction
PCR Labeling: Labeling a gene probe with DIG uses PCR.
Labeling of Probes Random-Primed Labeling: Gene probes, cloned or
PCR-amplified, and oligonucleotide probes are labeled
with radioactive isotopes and non-radioactive labels.
Nick translation
Radioactive Probes
Radioactive labeling
Most commonly involving enzymatic incorporation
of 32P, 33P, or 35S.
Radioactive labeling provides the most sensitive method
for detection, allowing detection of 0.01 pg.
Non-Radioactive Probes
A. Indirect non-radioactive labeling
Involve attaching the probe to an antibody directed against
an enzyme.
The immobilized probe is detected with enzyme, which
breaks down a chemiluminescent or chemifluorescent
substrate to produce a light signal.
Alternatively the probe can be labeled with biotin, making it
detectable by a streptavidin/avidin-enzyme conjugate.
B. Direct non-radioactive labeling
Involves attaching the probe to an enzyme (e.g., alkaline
phosphatase or horseradish peroxidase)
Probe can be detected directly using a chemiluminescent
or chemifluorescent substrate to produce a light signal.
Probes may also be labeled with fluorophores for
fluorescent detection.
Radioactive isotopes
32P
35S
3H
Non-radioactive labels
Biotin
Digoxigenin
Fluorescent dye (FISH)
Non-Radioactive labels:
Radioactive labels: P32
P33
S35
Digoxigenin, Fluorochromes, Biotin
Stages in ISHStages in ISH Preparation of Material Choice of Probes Labeling FixationFixation Hybridization Detection
Must be fixed or freezed as fast as possible in order to avoid the
effects of RNase on the RNA.
The fixation methods must be chosen to balance
1) The accessibility of the probe to the target sequence,
2) Retention of maximal levels of cellular target DNA or RNA,
3) Maintenance of morphological details in the tissue.
Fixation
To increase penetrance the tissue with detergents or proteases, but these may lead to the loss of DNA and RNA
Leads to decreased sensitivity resulting due to increased cross-linking or loss of mRNA during embedding.
Good sensitivity,
Fixatives
Acetic acid-alcohol mixtures
Glutaraldehyde
Paraffin and formalin fixation
4% paraformaldehyde solution
Best probe penetration,
Because of extensive protein cross-linking the probe penetrance is low.,
Best RNA Retention
Sectioning: Cryostat (frozen) sections are commonly used.
Usually the thickness is about 10-15 microns. Paraffin or resin
sections can also be used but not recommended.
The disadvantage of this method is that tissue morphology is
compromised, but it allows tissue to be used both for in situ
hybridization and for DNA or RNA extractions
Stages in ISHStages in ISH Preparation of Material Choice of Probes Labeling Fixation HybridizationHybridization Detection
Three Major Steps
I. Pre-hybridization step: The tissue is prepared for the
hybridization.
II. Hybridization.
III. Post-hybridization step: The hybridization signal on the
tissue is made visible.
A. Preparing the tissue for hybridizationPreparing the tissue for hybridization
For DNA hybridization: RNA is removed from the
tissue
by using RNase.
For RNA hybridization: Solutions, chemicals and tools
must be made RNase-free to avoid RNase contamination.
B. AsetilationAsetilation Commonly used for RNA/RNA hybridization. The aim of the asetilation is to neutralize the positive
charges on the tissue. This prevents the non-specific
binding of the probe.
I. Pre-hybridization
D. Neutralization of the endogeneous enzymes.Neutralization of the endogeneous enzymes.
E. Prehybridization fixationPrehybridization fixation
C. PPermeabilization of the tissueermeabilization of the tissue
Aim is to ease penetration of the probe in to the cells.
Carried out by
Protease Application
HCl application
Detergent Application
Digestion of proteins enhances the permeability.
Acidic denaturation of the membranes
Decrease the surface tension and break down the membranes.
I. Pre-hybridization
Denaturization
DNA/DNA hybridization:
In order to break dsDNA into single strands
RNA hybridization:
Sometimes the ssRNA probes curl on itself and appropriate
nucleotids bind to each other to make a partial double strand. This
can be avoided by denaturization.
II. Hybridization
Temperature for hybridization with the probe DNA/DNA hybridization: 37°C RNA/RNA hybridization: 50-55°C
Hybridization solution contain Probe Formamide Dextran sulphate DNA or tRNS blocking SDS BSA Salts
For the specifity of the hybridization
Increases the hybridization ratio
Increases the penetration of the probe
Blocks the non-specific reactions
Regulate the ionic environment
All double-stranded nucleic acids - whether dsDNA,
dsRNA or RNA:DNA hybrids - have specific "melting
temperatures”
Melting temperature depends on
specific Guanine+cytosine content
Hybrids
Ionic strength of solution
Length of the sequences to be annealed
Tm decreases by about 1°C for every 1% of mismatched
base pairs.
RNA:RNA> DNA:RNA>dsDNA
The shorter the probe sequence, the lower the melting temperature
Tm = (4 x [G+C]) (2 x [A+T])°C
dsDNA molecule exposed to high temperature or very
alkaline conditions leads to denaturation.
Denaturation occurs by heating dsDNA at about 100°C =
212°F.
Renaturation occurs by the two single stands of DNA for a
prolonged period at 65°C = 149°F
Hybridization Time Stringency of hybridization is increased by increasing the
temperature of incubation
The rate of hybridization/annealing is maximal at about Tm -
25°C, and too high a temperature results in very slow annealing.
Hybridization time is extended to ensure complete reaction
These reactions are performed at 10-20°C below the Tm value.
III. Post-Hybridization washes
Washing removes the faulty paired hybrids from the tissue.
RNase wash: In the RNA/RNA hybridization unhybridized
probes tend to stick on the tissue. This causes non-specific
results. The RNase breaks down the unhybridized probe.
RNase only effects the single strand RNA, so it does not break
the hybridization complex.
Stages in ISHStages in ISH Preparation of Material Choice of Probes Labeling Fixation Hybridization DetectionDetection
DetectionA) Detection of Non-radioactive Probes:
Immunohistochemical Method: A primary antibody against the
label is used: For example; anti-digoxigenin. The next steps are
the same with immunohistochemical method.
Biotin Avidin Method: If the probe is labeled with biotin, then
avidin is used to visualise the complex.
Systems with direct signaling: Fluorochromes, Enzymes, and
metals (colloidal gold).
DetectionB) Detection of Radioactive Probes:
Autoradiography: A method of detecting radioactively labeled
molecules through exposure of an x-ray sensitive photographic
film.
Film autoradiography:
Hybridization slides are put on a specific rontgen film.
Film are sensitive to the radioactive label used for
hybridization when exposed for a certain time.
Hybridization signal is seen as dark areas on the film.
Film Autoradiography
GluR7
GluR7
GluR7
GluR6
GluR7 Exression in the Median Eminence
Mapping of the Distribution of Glutamate Receptor mRNA’s in the Hypothalamus
GnRH mRNA Expression in Hypothalamus
Dual in situ Hybridization – GnRH and GluR5
Cells are arrested in metaphase and treated to make them swell and then fixed on to surface, thereby fixing chrmosomes on to the slides
The cells are treated so that the chromosomal DNA is denatured in to single strands
DNA probes are flooded on to the slide
Excess probes are washed away
Probes hybridize only with complementary sequence, which is the site of the gene of interest on a particular chromosome
The DNA probes are either themselves fluorescently labeled or bind to the probe DNA so to detect the site of the gene of interest and visualized under fluorescence microscope
Expose tissue section to X-ray film or emulsion
Develop film or emulsion-coated slides
In situ Methods
CISH Labeled complementary DNA or RNA strand is used to
localize a specific DNA or RNA sequence in a tissue
specimen.
CISH methodology may be used to evaluate
Gene amplification,
Gene deletion,
Chromosome translocation, and
Chromosome number.
Chromogenic Chromogenic in situin situ hybridization hybridization
What CISH offers: Evaluation of gene status simultaneously with tissue
morphology Use of existing bright-field microscopy and techniques similar
to IHC Archivable and quantitative results
CISH utilizes conventional peroxidase or alkaline phosphatase
reactions visualized under a standard bright-field microscope,
and is applicable to formalin-fixed, paraffin-embedded (FFPE)
tissues, blood or bone marrow smears, metaphase chromosome
spreads, and fixed cells.
CISH detection of non-amplified HER2 gene status
A. and amplified HER2 gene status
B. in different breast cancer tissue samples at 40X magnification.
CSH Probe created with Subtraction Probe Technology, a
proprietary technology that produces highly specific
probes by reducing repetitive sequences found in human
DNA
Probes do not require the repetitive sequence blocking that
is common for traditional cytogenetics DNA probes
ProtocolProtocol
Day 1
DenaturationAddition of probe
Pepsin digestion
Heat TreatmentTissue preparation
Hybridization
Day 2
Stringent Wash
Immunodetection
Interpretation
The radioactive Pax6 antisense DNA probe binds only where Pax6 mRNA is present, and can be visualized by developing the photographic emulsion. The locations where the probe has bound, depicted here as green dots, show that the Pax6 message is expressed in both the presumptive lens ectoderm and the optic stalk, which forms the retina and optic nerve. (From Grindley et al. 1995; photograph courtesy of R. E. Hill.)
In situ hybridization showing the expression of the Pax6 gene in the developing mouse eye.
Whole mount in situ hybridization
A digoxigenin-labeled antisense probe hybridizes to a specific mRNA. Alkaline phosphatase-conjugated antibodies to digoxigenin recognize the digoxigenin-labeled probe. The enzyme is able to convert a colorless compound into a dark purple precipitate.
Schematic of the procedure.
Pax6 mRNA can be seen to accumulate in the roof of the brain region that will form the eyes, as well as in the ectoderm that will form lenses. More caudal expression of this gene in the nervous system is also seen. (After Li et al. 1994; photograph courtesy of O. Sundin.)
Paraffin embedded skin wound labeled for procollagen I using a riboprobe and non-isotopic detection methodsA.Biotinylated probeB.B. DIG labelled probeC.Biotinylated probe detected by tyramide amplificationD.DIG labeled probe detected with anti-DIG alkaline phosphatase
Autoradiograph of liver for procollagen I using riboprobe. A.&B. frozen sections; C. paraffin embedded tissues
Equipments needed Plastic coverslips
Plastic (or glass) staining jars
Humid chamber :
Incubator or water bath at 37 °C (sometimes 42 °C) .
Digital thermometer
Programmable temperature-controlled heating block
Water bath
Microbiology (classic target - 16S rRNA)
Pathology
Developmental biology
Karyotyping
Phylogenetic analysis
- (Detection of chromosomal aberrations)
- morphology and
population structure of microorganisms
Applications of In Situ Hybridization
- (pathogen profiling, abnormal gene expression)
- (gene expression profiling in
embryonic tissues)
Physical mapping (mapping clones on chromosomes and
direct assignment of mapped clones to chromosomal
regions associated with heterochromatin or euchromatin).
Detection and Identification of Genetic Diseases and
Cancer
Detection of the differences of genetic expressions
Detection of mRNA expression and localization