In situ Hybridization (ISH) Method of localizing, either mRNA within the cytoplasm or DNA within the...
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Transcript of In situ Hybridization (ISH) Method of localizing, either mRNA within the cytoplasm or DNA within the...
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
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
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
• 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
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).
• 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
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
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
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) .