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