In situ Hybridization (ISH)

Method of localizing, either mRNA within the

cytoplasm or DNA within the chromosomes, by

hybridizing the sequence of interest to a

complimentary strand of a nucleotide probe.

Nucleic acid hybridization is a fundamental tool in

molecular genetics. It takes advantage of the

complementary nature of double stranded DNA or RNA

to the DNA or even RNA to RNA.

Quantitative RNA analysis

Technique Advantage Disadvantage

In situ hybridization

Single cell analysis,In situ spatial analysis, single cell sensitivity

Time consumingSingle genes

Northern Blot Size and quantityLarge RNA amounts needed (10-20 µg), single genes

Quantitative real-time RT-PCR

Most quantitative method

Single genes, specific primers needed

Semi quantitativeRT-PCR

Relatively quantitative

same

Laser micro dissection& qRT-PCR

Cell specificitySamePoor RNA quality

Microarray expression analysis

Thousands of genes analyzed at the same time

Thousands of cells needed, needs verification

Procedure

Drea et al. Plant Methods 2005 1:8 doi:10.1186/1746-4811-1-8

Tissue Preparation

• Detergents: Triton, SDS (permeabilization)• Proteinase K (permeabilization)

• Enzyme neutralization: H2O2 for peroxidase, levamisole for alkaline phosphatase

• Acetylation: 0.25 % acetic anhydride in triethanolamine (neutralization of positive charges)

• HCl (protein extraction and denaturation of target sequence)

Effect of Fixation and Proteinase Digestion

2.5 % glutaraldehyde

BM: Non-radioactive in situ hybridization, 1996

4% paraformaldehyde

0.05% 0.02% 0.005% 0.002% Proteinase K

Spinal Cord; probe PLP mRNA

Procedure

Drea et al. Plant Methods 2005 1:8 doi:10.1186/1746-4811-1-8

Probes

• Oligonucleotides: • single stranded DNA (RNase resistant)• Short 20-50 bases (good tissue penetration)• Cover only part of the mRNA, but potentially highly specific

• Single stranded DNA (200-600 bases)• Produced by Reverse transcription of RNA or primer amplified

• Double stranded DNA• denaturation necessary• only one strand is specific• Less sensitive due to self hybridization

• RNA• RNA-RNA hybrids are very stable and RNase resistant

• Post hybridization digestion with RNase possible

Bond Strength

RNA-RNA > RNA-DNA > DNA-DNA

• Advantages of RNA probes:– RNA-RNA hybrids are very stable– Tissue can be digested with RNase (dsRNA is not digested) after the hybridization reducing the background

– Higher specific activity compared to oligonucleotides

– Strand-specific compared to dsDNA probes

• Advantages of oligonucleotide probes:– Better tissue penetration– Potentially more specific

Procedure

Drea et al. Plant Methods 2005 1:8 doi:10.1186/1746-4811-1-8

Probe Labeling

• Non-radioactive labeling

• Direct:– The use of a nucleotides containing a fluorophore.

• Indirect: - Chemical coupling of a modified reporter molecule. The reporter molecule can bind with high affinity to another ligand (Biotin, Digoxigenin).

Non-radioactive direct labeling

Non-radioactive indirect labeling

• Biotin-streptavidin– Biotin is a naturally occuring vitamin which binds with high affinity (10-14). Highest known interaction in biology.

• Digoxigenin– A plant steroid which has a very specific antibody

Non-radioactive indirect labeling

Radioactive indirect labeling

Advantage: sensitivity

Disadvantage: hazard, long exposure times

– S35 medium half-life, good resolution

– P33 shorter half-life, good resolution– P32 short half-life, strong signal, bad resolution

– H3 long half-life, weak signal/quenching/long exposure times, good cellular resolution

Comparison of Labels

Radioactive Antigenic (non-radioactive)

Cost Frequent renewal

lower

Availability periodically continuous

Storage of label

short long

Duration of the protocol

Long (exposure time)

rapid

Storage of probe

short long

Sensitivity high Limited (better with TSA amplification)

Quantification

possible very difficult

Probe labeling

• Random primed labeling• PCR• In vitro transcription

Probe Labeling

• Random prime Labeling

In vitro Transcription

• Plasmid with T3, T7 or SP6 promoters• Linearization of plasmid DNA by restriction enzyme

• In vitro transcription: PlasmidbufferNTPlabeled UTPRNA polymerase

• DNAse digestion, Phenol/Chloroform extraction and RNA precipitataion

In vitro Transcription

T7

T3

EcoRI

BamHI

Antisense:Cut with EcoRIUse T-3 polymerase

Sense:Cut with BamHIUse T-7 polymerase

Procedure

Drea et al. Plant Methods 2005 1:8 doi:10.1186/1746-4811-1-8

Factors Influencing Hybridization

• Strand length– The longer the probe the more stable the duplex

• Base Composition– The % G:C base pairs are more stable than A:T

• Chemical environment– The concentration of Na+ ions stablize– Chemical denaturants (formamide or urea) destablize hydrogen bonds.

– Stringency of washes: temperature, salt concentration

Controls• Specificity of probe

– Sequence analysis– Testing by Northern blot

• Negative controls:– RNase treatment pre-hybridization– Addition of an excess of unlabeled probe– Hybridization with sense probe– Tissue known not to express the gene of interest

• Positive Controls:– Comparison with protein product– Comparison to probes hybridizing to different part of the same mRNA

– Tissue known to express the gene of interest– Poly dT probe or housekeeping gene to check RNA integrity

Ref: Anne Ephrussi, Daniel St Johnston, 2004, Cell, 116 (2), pages 143-152, 23 Jan

Multiplex mRNA detection

http://superfly.ucsd.edu/%7Edavek/images/quad.html

FISH

Clinical Applications of FISH

1. Characterization of chromosomal translocations

2. Aneuploidy analysis

3. Cancer specific chromosome deletions

FISH analysis -- translocation

mti-n.mti.uni-jena.de/~huwww/ MOL_ZYTO/imageAU9.JPG

Green + RED = YELLOW

metaphase

Pre-metaphase• Adapted from Albertson et al 2003 Nature Genetics 34:369-376

FISH + + + + + + +

acute promyelocytic leukemia

Aneuploidy revealed by FISH

http://68.33.28.8/geneticsweb/fish.htm

8 copies of chromosome 13

in pancreatic carcinoma Chromosome13-specific probe painting

http://lambertlab.uams.edu/images/cell.jpg

Interphase FISH, relaxed chromatin

Two green, two reds on different chromosomes

– no deletionTwo green, one red –One red is deleted.

GREEN SIGNAL SERVE AS A CONTROL PROBEON A SAME CHROMOSOME.

FISH analysis -- deletion

PRINS-PRimed In Situ labeling

• Alternative method for the identification of chromosomes in metaphase spreads or interphase nuclei.

• Denatured DNA is hybridized to short DNA fragments, or oligonucleotides followed by primer extension with labeled nucleotides.

• Labeling is detected with a fluorescent conjugated antibody.

• Limited sensitivity • rapid and low background staining.• Technique can be coupled with PCR (Cycling PRINS)  .