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 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

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% 0.002% Proteinase K 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 (200-600 bases) 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: 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).

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 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 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: 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 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

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 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

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. 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)  .