In Situ Polymerase Chain Reaction and Hybridization to Detect Low‐Abundance Nucleic Acid Targets

Omar Bagasra1

1 South Carolina Center for Biotechnology, Claflin University, Orangeburg, South Carolina
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.8
DOI:  10.1002/0471142727.mb1408s82
Online Posting Date:  April, 2008
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Abstract

This unit provides detailed methods and material descriptions for in situ hybridization following in situ amplification of DNA or RNA by PCR. It includes all essential components of the techniques, including variations suitable for different kinds of tissue and cell preparations. Planning, controls, and critical parameters for the amplification steps are discussed. Curr. Protoc. Mol. Biol. 82:14.8.1‐14.8.28. © 2008 by John Wiley & Sons, Inc.

Keywords: in situ hybridization; PCR; RT‐PCR; probes; primers; amplification; chambers

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: In Situ PCR Amplification of DNA and RNA Targets
  • Alternate Protocol 1: One‐Step Reverse Transcription and Amplification
  • Basic Protocol 2: Hybridization and Detection of ISPCR‐Amplified Target Material
  • Support Protocol 1: Preparation of AES‐Treated Slides
  • Support Protocol 2: Preparation of Specimens on Slides for ISPCR
  • Support Protocol 3: Labeling Oligonucleotide Probes Using 33P
  • Support Protocol 4: Optimization of Proteinase K Digestion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Situ PCR Amplification of DNA and RNA Targets

  Materials
  • Slides containing fixed specimens (see protocol 5)
  • 2% (w/v) paraformaldehyde in PBS, pH 7.4 (see recipe)
  • 1× and 3× PBS (see appendix 22)
  • 0.3% H 2O 2 in PBS (prepare fresh from 30% H 2O 2 stock)
  • 1 mg/ml proteinase K (Sigma; store in aliquots at −20°C)
  • RNase‐free DNase solution (see recipe)
  • Rinse solution: RNase‐free DNase solution (see recipe) without DNase
  • DEPC‐treated H 2O (unit 4.1)
  • 10× AMV/MoMuLV reaction buffer (see recipe)
  • 10 mM 4dNTP mix: 10 mM each dNTP in TE buffer, pH 7.5 (see appendix 22 for TE buffer; store 4dNTP mix up to 2 years at −20°C)
  • 40 U/µl RNasin (Promega)
  • 20 µM downstream primer (for reverse transcription; see )
  • 20 U/µl avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (MoMuLV) reverse transcriptase or 20 U/µl SuperScript II (with 5× reaction buffer; Invitrogen)
  • 0.1 M DTT
  • 25 µM forward and reverse primers (for PCR; see and unit 15.1)
  • 1 M Tris·Cl, pH 8.3 ( appendix 22)
  • 1 M KCl ( appendix 22)
  • 100 mM MgCl 2
  • 5 U/µl Taq DNA polymerase
  • 100% ethanol (optional)
  • 2× SSC ( appendix 22)
  • Frame‐Seal Incubation Chambers with plastic coverslips (Bio‐Rad)
  • 48°C (optional), 55°C (optional), 92°C, 95°C, and 105°C heating blocks accommodating glass slides
  • Coplin jars or glass staining dishes
  • Microscope with 400× magnification
  • Moist chamber (unit 14.2)
  • 37°C or 42°C incubator (optional)
  • Thermal cycler accommodating glass slides (see )

Alternate Protocol 1: One‐Step Reverse Transcription and Amplification

  • 100 µM forward and reverse primers (see and unit 15.1)
  • 3 mM 4dNTP mix: 3 mM each dNTP in TE buffer, pH 7.5 (see appendix 22 for TE buffer; store 4dNTP mix at −20°C)
  • 10 mM MnCl 2
  • 25 mM MgCl 2
  • 10× rTth transcription buffer: 100 mM Tris·Cl, pH 8.3 ( appendix 22)/900 mM KCl
  • 10× chelating buffer (see recipe)
  • 1.7 mg/ml BSA
  • 2.5 U/µl rTth DNA polymerase (Perkin‐Elmer or Invitrogen)

Basic Protocol 2: Hybridization and Detection of ISPCR‐Amplified Target Material

  Materials
  • 200 pM probe, 33P‐labeled (see protocol 6), fluorescently labeled, or biotin‐ or digoxigenin‐labeled (unit 3.18)
  • Deionized formamide
  • 20× SSC ( appendix 22)
  • 50× Denhardt's solution ( appendix 22)
  • 10 mg/ml sonicated salmon sperm DNA (unit 2.10), denatured (10 min at 94°C) before use
  • 10% (w/v) SDS
  • Frame‐Seal Incubation Chambers (with plastic coverslip; Bio‐Rad) containing ISPCR‐amplified nucleic acids (see protocol 1 or protocol 2)
  • Reagents for 33P detection:
    • 2× SSC ( appendix 22)
    • Diluted Kodak emulsion (unit 14.4)
    • Kodak D19 developer
    • Kodak Unifix fixer
    • 2% Gills hematoxylin (Sigma)
    • 1:1 (v/v) glycerol/PBS
  • Reagents for fluorescence detection:
    • PBS ( appendix 22)
    • 1:1 (v/v) glycerol/PBS ( appendix 22)/antifading reagent, e.g., 2% (w/v) Ficoll/ or 4% (w/v) n‐propyl gallate
  • Reagents for biotin‐labeled peroxidase‐based detection:
    • PBS ( appendix 22)
    • Streptavidin‐peroxidase conjugate working solution (see recipe)
    • AEC working solution (see recipe)
    • 2% Gills hematoxylin (Sigma)
  • Reagents for biotin‐labeled alkaline‐phosphatase‐based detection:
    • 2× SSC ( appendix 22)
    • Blocking solution (see recipe)
    • Streptavidin‐alkaline phosphatase conjugate working solution (see recipe)
    • 100 mM Tris·Cl, pH 7.5 ( appendix 22)/150 mM NaCl
    • Alkaline phosphatase substrate buffer (see recipe)
    • 75 mg/ml nitroblue tetrazolium (NBT) in 70% (v/v) dimethylformamide, freshly prepared
    • 50 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl phosphate (BCIP) in 100% dimethylformamide, freshly prepared
    • 1% nuclear fast red stain (Sigma)
  • Reagents for digoxigenin‐labeled peroxidase detection
    • PBS ( appendix 22)
    • Blocking solution (see recipe)
    • 1:250 peroxidase‐conjugated anti‐digoxigenin antibody (e.g., Sigma) in PBS
    • AEC working solution (see recipe)
    • 2% Gills hematoxylin (Sigma)
  • 50%, 70%, 90%, and 100% (v/v) ethanol (for color‐based methods)
  • Permanent mounting medium (for color‐based methods), water‐based (e.g., CrystalMount, Stephens Scientific or GelMount, Biomeda) or organic‐based (e.g., Permount, Fisher)
  • 95°C heating block accommodating glass slides
  • 48°C incubator
  • Moist chamber (see unit 14.2)
  • Light‐tight slide box with desiccant (for 33P detection)
  • Coplin jars or glass staining dishes
  • 20 × 60–mm glass coverslips
  • Microscope (for detecting biotin‐based probes) with fluorescence capability and appropriate filter sets (for detecting fluorescent probes)
NOTE: All washes and incubations are performed at room temperature unless otherwise noted.

Support Protocol 1: Preparation of AES‐Treated Slides

  Materials
  • 3‐aminopropyltriethoxysilane (AES; Sigma)
  • Acetone
  • DEPC‐treated H 2O (unit 4.1)
  • Coplin jars or glass staining dishes
  • Ultraclean glass slides
  • Vessel accommodating 1000 ml of liquid

Support Protocol 2: Preparation of Specimens on Slides for ISPCR

  Materials
  • 2 µM oligonucleotide probe (see )
  • 10 U/µl T4 polynucleotide kinase and 10× buffer (unit 3.10)
  • 10 µCi/µl[γ‐33P]ATP (10 Ci/mmol; Amersham)
  • TE buffer, pH 7.4 ( appendix 22)
  • 0.8‐ml Sephadex G‐50 column (e.g., QuickSpin, Boehringer Mannheim)
  • Additional reagents and equipment for labeling with T4 polynucleotide kinase (units 3.4& 3.10)

Support Protocol 3: Labeling Oligonucleotide Probes Using 33P

  Materials
  • Slides containing fixed specimens (see protocol 5)
  • 1 mg/ml proteinase K
  • 2% Gills hematoxylin (Sigma; or other appropriate stain)
  • 95°C heating block
  • High‐powered microscope
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Figures

Videos

Literature Cited

   Bagasra, O. and Hansen, J. 1997. In Situ PCR Techniques. John Wiley & Sons, New York.
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Key Reference
   Bagasra, O. and Hansen, J. 1997. In Situ PCR Techniques. John Wiley & Sons, New York.
  Describes the basic in situ amplification methods and discusses their applications.
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