Non‐isotopic RNA In Situ Hybridization on Embryonic Sections

Yacine Touahri1, Lata Adnani1, Pierre Mattar2, Kathryn Markham1, Natalia Klenin1, Carol Schuurmans1

1 Department of Biochemistry and Molecular Biology, Hotchkiss Brain Institute, Alberta Children's Hospital Research Institute, University of Calgary, Calgary, Alberta, 2 Current address: Cellular Neurobiology Research Unit, Institut de Recherches Cliniques de Montréal, Montréal, Quebec
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.22
DOI:  10.1002/0471142301.ns0122s70
Online Posting Date:  January, 2015
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Abstract

This unit describes methods for non‐isotopic RNA in situ hybridization on embryonic mouse sections. These methods can be used to follow the spatiotemporal dynamics of gene expression in an embryonic tissue of interest. They involve the use of labeled (e.g., digoxygenin, FITC) antisense riboprobes that hybridize to a specific mRNA in the target tissue. The probes are detected using an alkaline phosphatase−conjugated antibody recognizing the label and a chromogenic substrate. This method can be used to: (1) assess the expression of a single gene within a tissue, (2) compare the expression profiles of two genes within a tissue, or (3) compare the distribution of a transcript and protein within a tissue. While this approach is not quantitative, it provides a qualitative assessment of the precise cell types where a gene is expressed, which is not easily achievable with other more quantitative methods such as quantitative PCR. © 2015 by John Wiley & Sons, Inc.

Keywords: mRNA detection; in situ hybridization; embryonic mouse; spatiotemporal dynamics; gene expression

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: RNA In Situ Hybridization
  • Basic Protocol 2: Double RNA In Situ Hybridization
  • Basic Protocol 3: RNA In Situ Hybridization with Immunostaining
  • Support Protocol 1: Tissue Preparation and Sectioning
  • Support Protocol 2: Design and Generation of Digoxgenin‐Labeled Riboprobes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: RNA In Situ Hybridization

  Materials
  • RNase AWAY (Fisher Scientific)
  • DEPC‐treated water (see recipe)
  • Sigmacote (Sigma)
  • Chloroform
  • Frozen tissue sections (see protocol 4) in slide box at −80°C
  • 4% (w/v) formaldehyde (DEPC) (see recipe)
  • 1× PBS (DEPC) (see recipe)
  • TEA solution (DEPC) (see recipe)
  • Acetic anhydride
  • 2× SSC (DEPC) (see recipe)
  • 50% (v/v) formamide/1× SSC (DEPC)
  • Ethanol: 100% (Commercial Alcohols), and 50% and 70% (v/v) in DEPC‐treated water
  • Digoxygenin (DIG)−labeled hybridization probe (see protocol 5) at −20°C
  • Hybridization buffer (DEPC) (see recipe)
  • 5× SSC/50% (v/v) formamide or 1× salts (see recipe)/50% formamide
  • Washing solution (see recipe)
  • 1× MABT (see recipe)
  • RNase A and RNase A buffer (see reciperecipes; optional)
  • Blocking solution (see recipe)
  • Alkaline phosphatase (AP)−conjugated anti‐digoxygenin (anti‐DIG, Fab fragments version 16, Roche)
  • NTMT (see recipe) with and without 0.5 mg/ml levamisole
  • BCIP/NBT staining solution (see recipe)
  • Permount (Fisher Scientific)
  • Cover glass (Fisher Scientific)
  • 23 × 23−cm culture dish (Sarstedt)
  • 1‐ml pipets (Sarstedt)
  • Glass staining dishes with slide trays (Wheaton)
  • 3MM chromatography paper (Whatman)
  • Heating block(s) at 37°, 65°, and 95°C (Fisher)
  • 65°C water bath (e.g., Fisher Scientific)
  • Maker II Superfrost Marking Pens (Aspen Surgical)
  • 50‐ml Falcon tubes (VWR)
  • Gyrotory shaker (e.g., model G2, New Brunswick Scientific Co.)
  • Aluminum foil

Basic Protocol 2: Double RNA In Situ Hybridization

  Materials
  • Reagents and equipment for RNA in situ hybridization (see protocol 1)
  • FITC‐labeled hybridization probe (see protocol 5) at −20°C
  • 0.1 M glycine‐HCl, pH 2.2 (see recipe)
  • Alkaline phosphatase (AP)−conjugated anti‐FITC IgG (Sigma)
  • BCIP/INT staining solution (see recipe)

Basic Protocol 3: RNA In Situ Hybridization with Immunostaining

  Materials
  • Reagents and equipment for RNA in situ hybridization (see protocol 1)
  • Primary antibody to protein of interest
  • Secondary antibody conjugated to fluorophore of choice
  • PBT: 1× PBS containing 0.1% Triton X‐100

Support Protocol 1: Tissue Preparation and Sectioning

  Materials
  • Mouse embryos
  • 1× PBS (DEPC) (see recipe)
  • 4% (w/v) PFA (DEPC) (see recipe)
  • 20% (w/v) sucrose (DEPC) (see recipe)
  • Clear frozen section compound (O.C.T., VWR)
  • Dry Ice
  • Cryomolds
  • Cryostat (Leica)
  • Superfrost Plus slides (VWR)

Support Protocol 2: Design and Generation of Digoxgenin‐Labeled Riboprobes

  Materials
  • Plasmid containing cDNA for gene of interest
  • Restriction enzyme(s) with 10× buffers (Invitrogen or New England Biolabs)
  • 1 mg/ml bovine serum albumin (BSA, Sigma, optional)
  • DEPC‐treated water (see recipe)
  • 1% agarose gel
  • 1 kb+ DNA Ladder (Invitrogen; prepare according to manufacturer's instructions)
  • 6× loading dye (see recipe)
  • 1× TBE running buffer (see recipe)
  • 1:1 (v/v) phenol/chloroform (Amresco):
    • with Tris‐saturated phenol (pH 8.0) for DNA
    • with water‐saturated phenol (pH 4.5) for RNA
  • Chloroform
  • 3 M sodium acetate (NaOAc), pH 5.2 (see recipe)
  • Ethanol: 100% (Commercial Alcohols) and 75% (v/v) DEPC‐treated water
  • TE buffer (DEPC) ( appendix 2A)
  • 10× DIG RNA labeling mix, version 22 (Roche)
  • SP6, T7, or T3 polymerase with appropriate 10× or 5× transcription buffer (Invitrogen)
  • RNAsin (Promega)
  • In vitro transcription primer:
    • T3: CGC AAT TAA CCC TCA CTA AAG
    • T7: TAA TAC GAC TCA CTA TAG GG
    • SP6: GAT TTA GGT GAC ACT ATA G
  • DNase I (RNase‐free, New England Biolabs)
  • 0.2 M EDTA (DEPC), pH 8.0 ( appendix 2A)
  • 4 M LiCl in DEPC‐treated water
  • 0.2 N NaOH (DEPC) (see recipe)
  • 1 M dithiothreitol (DEPC) (DTT; store at −20°C)
  • 7.5 M ammonium acetate (NH 4OAc; see recipe)
  • Hybridization buffer (DEPC) (see recipe)
  • 1.5‐ml RNase‐free microcentrifuge tubes (Axygen)
  • Pipet tips with filters (Sarstedt)
  • Gel apparatus and power source (e.g., Bio‐Rad)
  • 37°C heating block (Fisher)
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Figures

Videos

Literature Cited

Literature Cited
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