Detection of mRNA expression and localization by yOm8H3


									           Advanced Research Techniques
           In Basic Medical Sciences

           “ In Situ ”
     Associate Professor Dr.
          Özhan Eyigör
Uludag University College of Medicine
Department of Histology & Embryology

• 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 –
• 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.
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

C) Permeabilization of the tissue:
   The aim is to ease the penetration of the probe into the
        1. Protease application: Use of the enzymes which
   digest the proteins enhances the permeability.
        2. HCl application: Acidic denaturation of the
        3. Detergent application: To decrease the surface
   tension and break down the membranes.

D) Neutralization of the endogeneous enzymes.

E) Prehybridization fixation

• 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 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
       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:
                Fluorochromes, Biotin

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 :


   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
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.

The DNA or RNA is hybridized by a complementary

The probe is labeled with radioactive or non-
  radioactive labels.

Following the hybridization the signal is made
      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
          Results are taken so fast

        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
       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.

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 (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
• 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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