1. 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.

2. Quantitative RNA analysis
Technique
Advantage
Disadvantage
In situ hybridization
Single cell analysis,
In situ spatial analysis, single cell sensitivity
Time consuming
Single genes
Northern Blot
Size and quantity
Large RNA amounts needed (10-20 µg), single genes
Quantitative real-time RT-PCR
Most quantitative method
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 analyzed at the same time
Thousands of cells needed, needs verification

3. Procedure
Drea et al. Plant Methods 2005
1:8 doi: /

4. 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)

5. Effect of Fixation and Proteinase Digestion4%
paraformaldehyde
2.5 %
glutaraldehyde
0.05% % % %
Proteinase K
Spinal Cord; probe PLP mRNA
BM: Non-radioactive in situ hybridization, 1996

6. Procedure
Drea et al. Plant Methods 2005
1:8 doi: /

7. Probes Oligonucleotides: Single stranded DNA (200-600 bases)
single stranded DNA (RNase resistant)
Short bases (good tissue penetration)
Cover only part of the mRNA, but potentially highly specific
Single stranded DNA ( 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

8. Bond Strength
RNA-RNA > RNA-DNA > DNA-DNA

9. 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

10. Procedure
Drea et al. Plant Methods 2005
1:8 doi: /

11. Probe Labeling Non-radioactive labeling Direct: Indirect:
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).

12. Non-radioactive direct labeling

13. Non-radioactive indirect labeling

14. 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

17. 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

18. 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
Sensitivity
high
Limited (better with TSA amplification)
Quantification
possible
very difficult

19. Probe labeling
Random primed labeling
PCR
In vitro transcription

20. Probe Labeling
Random prime
Labeling

21. 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

22. In vitro Transcription
Antisense:
Cut with EcoRI
Use T-3 polymerase
EcoRI T7
BamHI T3
Sense:
Cut with BamHI
Use T-7 polymerase

23. Procedure
Drea et al. Plant Methods 2005
1:8 doi: /

24. 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

25. Controls Specificity of probe Negative controls: Positive Controls:
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

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

28. Multiplex mRNA detection

29. FISH

30. Clinical Applications of FISH
Characterization of chromosomal translocations
2. Aneuploidy analysis
3. Cancer specific chromosome deletions

31. FISH analysis -- translocation
metaphase
FISH
Adapted from Albertson et al 2003 Nature Genetics 34:
Pre-metaphase
acute promyelocytic leukemia
Green + RED = YELLOW
mti-n.mti.uni-jena.de/~huwww/ MOL_ZYTO/imageAU9.JPG

32. Aneuploidy revealed by FISH
8 copies
of chromosome 13
in pancreatic carcinoma
Chromosome13-specific
probe painting

33. on different chromosomes
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.

34. 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)  .