Advanced Research Techniques In Basic Medical Sciences “ In Situ ” Hybridization Techniques Associate Professor Dr. Özhan Eyigör Uludag University College of Medicine Department of Histology & Embryology Hybridization • Hybridization: In solution – in vitro On cell preparations or tissue sections – in situ On nitrocellulose membranes • Southern Blotting: DNA • Northern Blotting: RNA What is In Situ Hybridization • In situ hybridization is a powerful technique that enables the detection of specific mRNA species within individual cells in tissue sections and can provide invaluable insights into physiological processes and disease pathogenesis. – Preservation of target mRNA ‘In Situ’ Hibridizasyon (hibridizasyon histokimyası, sitolojik hibridizasyon) • Morphologic demonstration of specific RNA (or DNA) sequences on: • Tissues • Cell cultures • Chromosome prepapations • In theory: The principle of the technique depends on the hybridization of labeled single-strand DNA (or RNA) sequences (= probe =complimentary secuence) to the cellular RNA or DNA. • 1969: Buongiorno-Nardelli and Amaldi – Nature • 1969: Gall and Pardue – PNAS • 1969: John, Birnstiel and Jones - Nature Usage of In situ Hybridization • Localization of the DNA sequences • Bacterial or viral DNA or RNA detection • Producing chromozomal genetic maps • Analysis of chromozomal errors • Detection of the differences of genetic expressions • Detection of mRNA expression and localization • Detection of virus or bacteria in tissues • Developmental biology • Cell biology • Histology • Genetics • Microbiology • Pathology Types of In situ Hybridization • Radioactive in situ hybridization : • Sulphur 35 (S35) • Phosphor 32, 33 (P32, P33) • Non-Radioactive in situ hybridization • Digoxigenin • Biotin • Dinitrophenil • Fluorochroms The Aim • To cause least possible damage on the tissue, • Not to block the probe to pass into the cell, • To preserve the RNA or DNA, • To avoid non-specific binding as possible, • To prevent contamination (especially RNase) Tissue Preparation • The tissue must be taken out of the animal as fast as possible to avoid the break down of the RNA or DNA. • Must be fixed or freezed as fast as possible in order to avoid the effects of RNase on the RNA. • Sectioning: Cryostat (frozen) sections are commonly used. Usually the thickness is about 10-15 microns. Paraffin or resin sections can also be used but not recommended. Hybridization Protocol Three Major Steps: • Pre-hybridization step: The tissue is prepared for the hybridization. • Hybridization. • Post-hybridization step: The hybridization signal on the tissue is made visible. Prehybridization A) Preparing the tissue for hybridization For DNA hybridization: RNA is removed from the tissue by using RNase. For RNA hybridization: The solutions, chemicals ant the tools must be made RNase-free to avoid RNase contamination. B) Asetilation Commonly used for RNA/RNA hybridization. The aim of the asetilation is to neutralize the positive charges on the tissue. This prevents the non-specific binding of the probe. Prehybridization C) Permeabilization of the tissue: The aim is to ease the penetration of the probe into the cells. 1. Protease application: Use of the enzymes which digest the proteins enhances the permeability. 2. HCl application: Acidic denaturation of the membranes 3. Detergent application: To decrease the surface tension and break down the membranes. D) Neutralization of the endogeneous enzymes. E) Prehybridization fixation Denaturization • In order to break the double stranded DNA into single strands for DNA/DNA hybridization. • Also used for RNA hybridization: Sometimes the single-stranded RNA probes curl on itself and appropriate nucleotids bind to each other to make a partial double strand. This can be avoided by denaturization. Hybridization • Hybridization with the probe. • For DNA/DNA hybrids at 370 C • For RNA/RNA hybrids at 50-550 C • The concentration of the probe must be determined. • The contents of the hybridization solution: 1. Probe 2. Formamide (For the specifity of the hybridization) 3. Dextran sulphate (Increases the hybridization ratio) 4. Blocking DNA or tRNA (somon sperm DNA) 5. Sodium dodesyl sulphate (Increases the penetration of the probe) 6. Bovine serum albumine (Blocks the non-specific reactions) 7. Salts (to regulate the ionic environment) Post-Hybridization washes • The stringency of the in situ hybridization: Determines the ratio of the faultless hybridization of the probe with the target sequence. The temperature, ionic concentration and the amount of the formamide are important. Washing removes the faulty paired hybrids from the tissue. RNase wash: In the RNA/RNA hybridization unhybridized probes tend to stick on the tissue. This causes non-specific results. The RNase breaks down the unhybridized probe. RNase only effects the single strand RNA, so it does not break the hybridization complex. Preparation and Labeling of the Probe 1. Two strand DNA probes 2. Synthetic oligonucleotide probes. 3. Single strand complementary DNA (cDNA) probes. 4. Complementary RNA (cRNA) probes. *RNA polymerase One polymerase works on one direction: Antisense Probe Other polymerase works on the other direction: Sense Probe Antisense probe is used for the hybridization. Sense probe has the same sequence with the target nucleotide strand. *Labeled nucleotid (UTP) *Labeled probe Non-Radioactive labels: Digoxigenin, Fluorochromes, Biotin Radioactive labels: P32, P33, S35 Detection of Hybridization A) Detection of Non-radioactive Probes: Immunohistochemical Method: A primary antibody against the label is used: For example; anti-digoxigenin. The next steps are the same with immunohistochemical method. Biotin Avidin Method: If the probe is labeled with biotin, then avidin is used to visualise the complex. Systems with direct signaling: Fluorochromes, Enzymes, and metals (colloidal gold). Detection of Hybridization B) Detection of Non-radioactive Probes : Autoradiography: 1. Film Autoradiography : Hybridization slides are put on a specific rontgen film. This film must be sensitive to the radioactive label used for hybridization. The film should be exposed for a certain time. After developing the hybridization signal is seen as dark areas on the film. Film Autoradiography Detection of Hybridization B) Detection of Non-radioactive Probes : 2. Autoradiography on slides: After hybridization the slides are covered with a photographic emulsion. Exposed for a certain time. After development the hybridization signal is seen as black dots on the section. When analyzed with a dark field condenser the signal is seen as white shiny dots over a dark background. GluR7 Exression in the Median Eminence GluR7 GluR7 GluR7 GluR6 Mapping of the Distribution of Glutamate Receptor mRNA’s in the Hypothalamus GnRH mRNA Expression in Hypothalamus Dual in situ Hybridization – GnRH and GluR5 FISH: Fluorecence in situ Hybridization Antisense Method – (Antisense treatment) •Southern and Northern Blotting Southern blotting determines the presence of a particular gene or DNA sequence within thousands of base pairs in a DNA molecule. It is widely used for the molecular biology and recombinant DNA technology studies. Especially used to determine the presence of a particular DNA in the studies of: Gene structure Gene expression Genomic organization Gene transfer Southern blotting was first introduced by Southern in 1975. Northern Blotting is used to determine RNA with slight modification of southern blotting technique. •Southern and Northern Blotting Sometimes it's a bit hard to understand, but there is humor in science. In the 1970s, E.M. Southern developed a method for locating a particular sequence of DNA within a complex mixture. This technique came to be known as Southern blotting. In a tongue-in-cheek fashion, those who used a similar method for locating a sequence of RNA named it Northern blotting. It is also known as Northern hybridization or RNA hybridization. Principle The DNA or RNA is hybridized by a complementary probe. The probe is labeled with radioactive or non- radioactive labels. Following the hybridization the signal is made visible: Radioactive label – autoradiography Biotin label – immunohistochemistry Flourescent label – flourescence microscopy Radioactive Labeling In the recent years most of the studies used radioactive labels. The advantages of radioactive labeling and autoradiography: Results are taken so fast Reliability Disadvantages: Not easy to use radioactivity It is dangerous Non-Radioactive Labeling 1. Digoxigenin-anti-digoxigenin system, 2. Horseradish peroksidaz system 3. Biotin-streptavidin system In order to visualise the complexes of these systems: The use of chromogens (colorimetric): Makes a colored precipitate. Use of chemiluminergic substances: Emit light to be observed under direct ultraviolet light or flourescence microscope. Technique The technique: • Similar to Western blotting. • DNA or RNA is seperated on a gel according to their size by electrophoresis • Transterred to a nitrocellulose membrane • Hybridization is employed on the nitrocellulose membrane • The hybridization signal is visualised. FISH • FISH (Fluorescent in situ hybridization) is a cytogenetic technique which can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes. • It uses fluorescent probes which bind only to those parts of the chromosome with which they show a high degree of sequence similarity. F • luorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome. • FISH is often used for finding specific features in DNA. These features can be used in genetic counseling, medicine, and species identification.
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