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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 .

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in situ hybridization ish
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.

procedure
Procedure

Drea et al. Plant Methods 2005

1:8 doi:10.1186/1746-4811-1-8

tissue preparation
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
Effect of Fixation and Proteinase Digestion

4%

paraformaldehyde

2.5 %

glutaraldehyde

0.05% 0.02% 0.005% 0.002%

Proteinase K

Spinal Cord; probe PLP mRNA

BM: Non-radioactive in situ hybridization, 1996

procedure6
Procedure

Drea et al. Plant Methods 2005

1:8 doi:10.1186/1746-4811-1-8

probes
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
Bond Strength

RNA-RNA > RNA-DNA > DNA-DNA

slide9
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
procedure10
Procedure

Drea et al. Plant Methods 2005

1:8 doi:10.1186/1746-4811-1-8

probe labeling
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 indirect labeling14
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
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
probe labeling19
Probe labeling
  • Random primed labeling
  • PCR
  • In vitro transcription
probe labeling20
Probe Labeling
  • Random prime

Labeling

in vitro transcription
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 transcription22
In vitro Transcription

Antisense:

Cut with EcoRI

Use T-3 polymerase

EcoRI

T7

BamHI

T3

Sense:

Cut with BamHI

Use T-7 polymerase

procedure23
Procedure

Drea et al. Plant Methods 2005

1:8 doi:10.1186/1746-4811-1-8

factors influencing hybridization
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
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
multiplex mrna detection
Multiplex mRNA detection

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

slide30

Clinical Applications of FISH

Characterization of chromosomal translocations

2. Aneuploidy analysis

3. Cancer specific chromosome deletions

fish analysis translocation
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
Aneuploidy revealed by FISH

8 copies

of chromosome 13

in pancreatic carcinoma

Chromosome13-specific

probe painting

http://68.33.28.8/geneticsweb/fish.htm

slide33

FISH analysis -- deletion

Two green, two reds

on different chromosomes

– no deletion

Two green, one red –

One red is deleted.

Interphase FISH, relaxed chromatin

GREEN SIGNAL SERVE AS A CONTROL PROBE

ON A SAME CHROMOSOME.

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

prins pr imed i n s itu labeling
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) .