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 consuming
Single genes
Size and quantity
Large RNA amounts
needed (10-20 µg), single
genes
Northern Blot
Quantitative real-time RTMost quantitative method
PCR
Single genes, specific
primers needed
Semi quantitative
RT-PCR
Relatively quantitative
same
Laser micro dissection
& qRT-PCR
Cell specificity
Same
Poor RNA quality
Microarray expression
analysis
Thousands of genes
Thousands of cells
analyzed at the same time 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
4%
paraformaldehyde
2.5 %
glutaraldehyde
0.05%
0.02%
0.005%
Proteinase K
0.002%
Spinal Cord; probe PLP mRNA
BM: Non-radioactive in situ hybridization, 1996
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
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
Radioactive indirect labeling
Advantage: sensitivity
Disadvantage: hazard, long exposure times
–
–
–
–
S35
P33
P32
H3
medium half-life, good resolution
shorter half-life, good resolution
short half-life, strong signal, bad resolution
long half-life, weak signal/quenching/long
exposure times, good cellular resolution
Comparison of Labels
Radioactive
Antigenic
(non-radioactive)
Cost
Availability
Storage of label
Frequent renewal
periodically
short
lower
continuous
long
Duration of the
protocol
Storage of probe
Sensitivity
Long (exposure
time)
short
high
rapid
Quantification
possible
long
Limited (better
with TSA
amplification)
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: Plasmid
buffer
NTP
labeled UTP
RNA polymerase
• DNAse digestion, Phenol/Chloroform extraction
and RNA precipitataion
In vitro Transcription
Antisense:
Cut with EcoRI
Use T-3 polymerase
EcoRI
T7
BamHI
T3
Sense:
Cut with BamHI
Use 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
, 23 Jan
Ref: Anne Ephrussi, Daniel St Johnston, 2004, Cell, 116 (2), pages 143-152
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
metaphase
FISH + + +
•
+
+
+
+
Adapted from Albertson et al 2003 Nature Genetics
34:369-376
Pre-metaphase
acute promyelocytic leukemia
Green + RED = YELLOW
mti-n.mti.uni-jena.de/~huwww/ MOL_ZYTO/imageAU9.JPG
Aneuploidy revealed by FISH
8 copies
of chromosome 13
in pancreatic carcinoma
Chromosome13-specific
probe painting
http://68.33.28.8/geneticsweb/fish.htm
FISH analysis -- deletion
Interphase FISH, relaxed chromatin
Two green, two reds
on different chromosomes
– no deletion
Two green, one red –
One red is deleted.
GREEN SIGNAL SERVE AS A CONTROL PROBE
ON A SAME CHROMOSOME.
http://lambertlab.uams.edu/images/cell.jpg
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) .