Introduction to In Situ Hybridization

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Introduction to In Situ Hybridization IGP
Methodology Fall 2002 Heath Acuff - Matrisian
Lab Elizabeth Forrester - Moses Lab Department of
Cancer Biology October 31, 2002
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General Background

• Method of localizing either mRNA within the
  cytoplasm or DNA within the chromosomes of the
  nucleus by hybridizing the sequence of interest
  to a complimentary strand a nucleotide probe
• Normal hybridization isolation, separation,
  blotting, probing
• In situ hybridization basic principles are the
  same, except utilizing the probe to detect
  specific nucleotide sequences WITHIN cells and
  tissues
• Sensitivity of 10-20 copies of mRNA or DNA per
  cell

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General Background

• In situ tells you the LOCATION of expression,
  NOT QUANTITY!!!!
• Unique set of problemscell/tissue permeability,
  which type of probe to choose, how to label it
• As many in situ methods/protocols as there are
  tissues for you to probe, so its more important
  to understand the different stages in the process
  and their purpose rather than having a recipe!!!!

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Materials and Preparation

• Most common tissue sections used are
• Frozen sections- Fresh tissue is snap frozen and
  then embedded into a special support medium for
  thin cryosectioning. Fixation occurs rapidly in
  4 PFA just prior to hybridization
• Paraffin embedded- Sections are fixed in formalin
  and embedded in wax before being sectioned
• Cells in Suspension- Cells are cytospun onto
  glass slides and fixed with methanol
• Metaphase Chromosomal Spreads- fixed with a
  mixture of methanol and acetic acid

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Permeablization

• Why???

• The probe has to reach its target, i.e the mRNA
  of the target gene located in your sample.

• Three common reagents used are
• 1) HCl- 0.2M for 20-30 min, extraction of
  proteins and hydrolysis of target sequence
  may help decrease the level of
  background staining
• 2) Detergent treatment- Trition X-100, SDS work
  by extracting lipids. Not
  usually required in tissue that
  has been embedded in wax
• 3) Proteinase K- non-specific
  endopeptidase, active over a wide pH and
  not easily inactivated. Removes protein
  that surrounds the target sequence. Starting
  point 1µg/ml but needs to be determined on
  individual basis.

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The Probe

• What is a probe anyway???

- complimentary sequences of nucleotide base to
specific mRNA sequencce of interest
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Types of Probes

• Esssentially FOUR TYPES used in in situ
  hybridizations

• OLIGONUCLEOTIDE PROBES
• - Produced synthetically and utilize readily
  available deoxynucleotides
• - Requires that you know the specific nucleotide
  sequence
• CANT JUST PICK ANY OL SEQUENCE WITHIN THE
  CODING REGION!!!!!!!!!!
• Resistant to RNases, generally around 40-50 bp
  making them ideal for in situ because of easy
  penetration into cells or tissue.
• G/C vs A/U content
• SINGLE STRANDED, ie cannot renature!

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Types of Probes

• 2) SINGLE-STRANDED DNA PROBES
• - Similar advantages to oligonucleotide probes
  except they are much LARGER!! (About 200-500bp)
• Produced by reverse transcription of RNA or by
  amplified primer extension of a PCR-generated
  fragment in presence of a single antisense
  fragment
• However, this is the disadvantage!! Time
  consuming to prepare, expensive reagents,
  extensive molecular biology skills to
  troubleshoot.

• 3) DOUBLE-STRANDED DNA PROBES
• Produced by the inclusion of your sequence of
  interest in bacteria, sequence is
  excised with restriction enzymes- if sequence is
  unknown
• If sequence is known, PCR!!
• Advantage large quantities of your probe
• Disadvantage must add denaturation step prior to
  hybridization and are generally less sensitive
  due to rehybridization

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Types of Probes

• 4) RNA PROBES
• - Stability is a BIG advantage. RNA-RNA hybrids
  are very thermostable and resistant to digestion
  by RNases
• - Digest with RNase post-hybridization to remove
  non-hybridized RNA, therefore less background
  staining
• - Two methods to prepare
• i) RNA polymerase-catalyzed txn of mRNA in 3
  to5 direction
• ii) In vitro txn of lineraized plasmid
  DNA with RNA polymerase, plasmid
  vectors containing polymerase from bacteriophages
  T3, T7 or SP6 are used
• - Disadvantage can be VERY difficult to work
  with since they are very sensitive to RNases, so
  require sterile technique
• - Most widely used probe

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Labeling The Probe

• 1)RADIOLABELING
• - 32P, 35S or 3H
• - Detection of transcripts at low levels
• - Incubation times up to several weeks!!!
• - Visualized by dipping slides into photographic
  emulsion and storing at -80oC in the dark
• - Develop as normal photograhic film
• View though developed emulsion, black silver
  grains indicate sites of labeled transcripts
• Hazardous to work with!!!
• Takes longer to get results

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Labeling The Probe

• 1) NON-RADIOLABELED
• biotin, digoxin, digoxigenin (DIG), alkaline
  phosphotase, fluorescent labels, fluorescein
  (FITC), Texas Red and rhodamine
• Anti-(DIG) antibodies not likely to bind to any
  other biological material
• Have no inherent decay kinetics
• Once labeled use immediately or divide into
  aliquots and store at -20oC for use up to 2 years
  later

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Labeling The Probe
There are MANY ways to label probes depending
on the task they are required for!

• Homogenous Labeling Methods for DNA probes
• - Random inc. of labeled nucleotides throughout
  the length of the probe
• - Three methods to randomly prime DNA
• i) Random primed- linearized template DNA is
  denatured and annealed to a primer. Klenow
  is used to synthsize new DNA staring at 3
  end of annealing primer in presence
  of labeled and unlabeled nucleotides
• ii) Nick translation- Nick one srand
  of DNA with Dnase I hen a 5-3 exonuclease,
  DNA pol I, extends the nicks to gaps and
  then the pol replaces excised nucleotides with
  labeled ones. Can get VERY STRONG labeling
  in short time(60-90m)
• iii) PCR- PCR using labeled
  nucleotides, large amounts of labeled probe
  with only minimal amounts of DNA (1-100pg)

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Labeling The Probe
2) Homogenous Labeling Methods for RNA
probes - In vitro transcription from a
linearized DNA template which contains a
promoter for RNA polymerase - SP6, T3 or T7 RNA
polymerases most commonly used - Takes about
1-2 hours - Produces about 10 fold amounts of
labeled RNA form 1 unit of DNA 3) Other
Methods - End-labeling of DNA probes- by
tagging on a labeled nucleotide to the 3 or
5 end of the probe. Enzyme based rxn so you
dont have to denature the DNA. 3 uses a
terminal transferase which attaches a single
labeled nucleotide to the 3-OH end. 5 uses a
polynucleotide kinase and obviously requires
the dephosphorylation of the DNA. Which do
you think is preferred???? - Advantage to
end-labeling is that it is unlikely to interfere
with hybridization and gives a shaper signal,
esp. with radioactive probes
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Hybridization
DNA must reanneal to a complimentary strand
just below the DNAs melting point
(Tm)--temperature at which half the DNA is
present in a single stranded or denatured form
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Factors that influence TM and renaturation of DNA

• Temperature
• pH (Buffer- pH 6.5-7.5 higher pH-more stringent
  hybridization)
• Conc. of monovalent cations (Na ions interact
  with phosphate groups of nucleic acids,
  decreasing electrostatic interactions between the
  two strands higher salt conc increases the
  stability of the hybrid)
• Presence of organic solvents (Formamide, reduce
  the melting temp of DNA-DNA and DNA-RNA duplexes
  so that hybridization can be performed at a lower
  temp)

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Additional factors affecting hybridization
probe length--influences thermal stability
probe concentration--higher the conc. of probe
the higher the reannealing rate dextran
sulfate--added to hybridization buffer strongly
hydrated, preventing macromolecules from having
access to water, increases probe conc. and
hybridization rates
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Following hybridization.
Stringency washes--remove background associated
with nonspecific hybridization, wash with a
dilute solution of salt, the lower the salt and
higher the wash temperature, the more stringent
the wash.
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Controls
Positive controls Negative controls Sense
probes-determine back ground signal, non-specific
binding Probe for house keeping sequences-
actin Competition studies with labeled and
unlabeled probes to demonstrate that binding is
specific
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Localization of DNIIR and TGFa transgenes in
Mammary Gland Tumors Arising from
MMTV-DNIIR/TGFa Transgenic Mice
T5 As DNIIR
T5 As DNIIR
T1 As TGFa
T5 As TGFa
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Localization of DNIIR transgene in MMTV-DNIIR
Transgenic Tumors
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Localization of DNIIR Transgene in Tumors Arising
From MMTV-DNIIR/TGF-a Transgenic Mice
T3 DNIIR/TGF-a As
T2 DNIIR/TGF-a As
T3 DNIIR/TGF-a Sense
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Methods of in situ hybridization
Radioactive-radiolabel probes--32P, 35S, 3H
Non-radioactive- direct and indirect FISH-
Fluorescence in situ hybridization In situ
PCR PRINS- PRimed IN Situ labeling TSA-
Tyramide Signal Amplification
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Fluorescence in situ hybridization (FISH)
Fluorescent molecules are used to identify genes
or chromosomes Locus specific probes- hybridize
to a particular region of a chromosome -
determine which chromosome a gene is located
on Alphoid or centromeric repeat probe-
generated from repetitive sequences found at
the centromeres of chromosomes - determine if an
individual has the correct number of
chromosomes Whole chromosome probes- collection
of smaller probes that hybridize to a
different sequence along the length of a
chromosome - determine chromosome abnormalities
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• Interphase nuclei from amniotic fluid cells
• Chromosome 18-aqua
• Chromosome x-green
• Chromosome y-red

• Metaphase cell--Two probes
• for chromosome 22
• Green- internal control
• Red- DiGeorge region of chromosome 22

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In situ PCR
Amplify a single copy of a gene or low levels of
mRNA using PCR Cells or tissues are
permiabalized and incubated with a solution
containing primers, nucleotides and polymerase
and subjected to PCR amplification. The
transcripts are visualized with standard in situ
hybridization techniques Caveats (1)
miss-priming-amplification of non-specific DNA
(2) non-specific incorporation of labeled
nucleotides through fragmented DNA which
undergoes repair (3) endogenous priming, priming
of non-specific PCR products by cDNA or DNA
fragments
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In situ PCR using DIG-labeled gag primers for the
detection of HIV DNA in bone marrow
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PRINS-Primed IN situ labeling
Identify chromosomes in metaphase spreads or
interphase nuclei
Denatured DNA is hybridized to short DNA
fragments or oligos and primer extension is then
carried out with polymerase and labeled
nucleotides. The labeled site is then detected
using a fluorescent antibody.
Fok I repeat detected by PRINS-green
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Tyramide Signal Amplification (TSA)
Biotin
Streptavidin-HRP complex
Biotinyl tyramide
DAB
Biotin-labeled probe
Probe hybridizes to the target nucleic acid
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Any Questions????
References
Non-radioactive In situ Hybridization Application
Manual, Boehringer Mannheim. 2nd Ed.
1996 http//martin.parasitology.mcgill.ca/insituh
ybridization/ insitu.htm Heath.Acuff_at_mcmail.vande
rbilt.edu Elizabeth.Hamilton_at_mcmail.vanderbil.edu
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HAPPY HALLOWEEN!!!!!