microRNA Profiling as Tool for Developmental Neurotoxicity Testing (DNT)

Lena Smirnova1, Andrea E.M. Seiler1, Andreas Luch2

1 Department of Experimental Toxicology and Center for Documentation and Evaluation of Alternatives to Animal Experiments (ZEBET), German Federal Institute for Risk Assessment (BfR), Berlin, 2 Department of Chemicals and Products Safety, German Federal Institute for Risk Assessment (BfR), Berlin
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.9
DOI:  10.1002/0471140856.tx2009s64
Online Posting Date:  May, 2015
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Abstract

microRNAs (miRNAs) are small non‐coding RNA molecules functioning as post‐transcriptional regulators of gene expression. miRNAs play a significant role in organism development, regulating developmental timing, cell differentiation, and specification. In the developing brain, miRNAs regulate neural stem cell differentiation, lineage specification, synaptogenesis, and brain morphogenesis. Temporal and spatial specificity of miRNA expression make them an attractive marker to study cellular responses to toxicant exposure. Neural differentiation of murine embryonic stem cells (mESCs) has been established as an alternative method to study developmental neurotoxicity (DNT) in vitro. This unit will describe a method for miRNA profiling (miRNomics) as a molecular end point to study developmental neurotoxicity. A protocol for neural differentiation of mESC will be described as a cellular model for DNT testing. The miRNomics protocol is versatile and can be used with other DNT cellular systems such as primary cultures, human embryonic stem cells (hESCs), or induced pluripotent stem cells (iPSCs). © 2015 by John Wiley & Sons, Inc.

Keywords: miRNA; miRNomics; in vitro developmental neurotoxicity; embryonic stem cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Neural Differentiation of mESCs
  • Support Protocol 1: Characterization of Neural Differentiation by Flow Cytometry and Immunocytochemistry
  • Support Protocol 2: Toxicant Treatment During Neural Differentiation
  • Basic Protocol 2: miRNA Profiling Using Affymetrix miRNA Microarray
  • Basic Protocol 3: Analysis of miRNA Expression by Two‐Step Quantitative RT‐PCR (qPCR)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Neural Differentiation of mESCs

  Materials
  • Murine embryonic stem cells (mESCs): cell line W4 (Prof. Rolf Kemler, Max Planck Institute, Freiburg, Germany) or alternatively cell line D3 (ATCC/LGC Standards GmbH, Wesel, Germany)
  • Routine culture medium (see recipe)
  • 0.1 mg/ml poly‐L‐ornithine (PLO; Sigma Aldrich, cat. no. 4957)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca2+ and Mg2+ (PAN Biotech, cat. no. P04‐36500)
  • N2B27 neural differentiation medium (see recipe)
  • TrypLE Express (Life Technologies)
  • PAN ES fetal calf serum (PANSERA, FCS; PAN‐Biotech)
  • 6‐cm tissue culture dishes (Becton Dickinson)
  • Flat‐bottomed tissue culture microtiter plates (6‐, 24‐, or 96‐well, Becton Dickinson)
  • Centrifuge
  • Phase‐contrast microscope
  • Additional reagents and equipment for mammalian cell tissue culture, including counting cells ( appendix 3B)

Support Protocol 1: Characterization of Neural Differentiation by Flow Cytometry and Immunocytochemistry

  Materials
  • Neural‐differentiated ESCs ( protocol 1) in 6‐well plates (for flow cytometry) or on coverslips in 24‐well plates (for immunocytochemistry)
  • PBS‐EDTA solution (see recipe)
  • Dissociation solution (see recipe)
  • PAN ES fetal calf serum (PANSERA, FCS; PAN‐Biotech)
  • Wash solution I (WS I; see recipe)
  • 4% paraformaldehyde (PFA; see recipe)
  • Blocking solution (see recipe)
  • Wash solution II (WS II; see recipe)
  • FACSFlow sheath fluid (BD Biosciences)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca2+ and Mg2+ (PAN‐Biotech, cat. no. P04‐36500)
  • Antibodies (see Table 20.9.1)
  • Hoechst 33342: dilute Hoechst 10 mg/ml stock solution (Invitrogen, cat. no. H3569) 1:10,000 in DPBS (store at 4°C)
  • Immunomount mounting medium (Dako, cat. no. S3023))
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Refrigerated centrifuge
  • 5‐ml polystyrene round‐bottom tubes with filter caps for flow cytometry (BD Biosciences)
  • Flow cytometer (e.g., FACSCantor or FACSCalibur; BD Biosciences)
  • Shaker
  • Microscope slides
  • Fluorescent microscope with appropriate filters
  • Additional reagents and equipment for mammalian cell tissue culture, including counting cells ( appendix 3B)
Table 0.9.1   MaterialsAntibody Dilutions

Antibody Flow cytometry Immunocytochemistry
Anti‐mouse MAP2 AB (clone Ap‐20, BD) 1: 800 1:1000
Anti‐mouse β‐III‐tubulin (clone SDL.3D10, Sigma Aldrich) 1:3200 1:3200
Anti‐mouse GFAP (clone G‐A‐5, Sigma Aldrich 1:1600 1:1600
RPE‐conjugated goat anti‐mouse secondary antibody (Dako) 1:60
Cy3/5 goat anti‐mouse secondary antibody (Dianova) 1:400

Support Protocol 2: Toxicant Treatment During Neural Differentiation

  Additional Materials (also see protocol 1)
  • Test chemical (toxicant) stock solutions and appropriate solvents
  • 1 mg/ml resazurin stock solution prepared in DPBS (Sigma‐Aldrich, cat. no. R7017‐1G); alternatively, CellTiter®‐Blue Cell Viability Assay (100‐ml; Promega) can be used
  • 8‐channel‐pipettors and dilution block
  • 96‐well flat‐bottom tissue culture microtiter plates, Optilux, black/clear bottom (Becton Dickinson)
  • Shaker
  • Fluorescence microplate reader with the facility to emit light at 560 nm (excitation) and to measure a fluorescent signal at 590 nm (emission)

Basic Protocol 2: miRNA Profiling Using Affymetrix miRNA Microarray

  Materials
  • TRIzol reagent (Life Technologies)
  • Neural‐differentiated ESCs ( protocol 1) in 6‐well plates)
  • RNaseZAP solution (Sigma‐Aldrich)
  • miRNeasy Mini Kit (Qiagen; optional)
  • mirVAna miRNA isolation kit (Life Technologies; optional)
  • RNase‐free DNase Set (Qiagen, cat. no. 79254)
  • Acid‐phenol:chloroform, pH 4.5 (with IAA, 125:24:1; Life Technologies)
  • Ethanol, absolute (100%; 200 Proof), molecular biology grade
  • 3 M sodium acetate, pH 5.2 (Cellgro; also see recipe in appendix 2A)
  • 75% ethanol
  • Isopropanol (>99.9%)
  • RNase‐free water
  • FlashTag Biotin RNA labeling kit (Genisphere)
  • Reagents for ELOSA QC assay (appendix a and c, Affymetrix FlashTag Biotin RNA labeling kit guide)
  • Phase‐lock‐gel tubes (5 PRIME)Refrigerated centrifuge
  • Nanodrop spectrophotometer ND‐1000 (Nanodrop Technologies)
  • Affymetrix GeneChip Command Console (AGCC) software
  • Affymetrix Expression Console software (EC) v. 1.2 or higher
  • GeneChip Scanner 3000 7 G (Affymetrix)
  • miRNA array chip (v. 4.0; Affymetrix)
  • GeneChip Fluidics Station 450 (Affymetrix)
  • GeneChip Hybridization Oven 645 (Affymetrix)
  • GeneChip eukaryotic hybridization control kit (Affymetrix)
  • GeneChip hybridization, wash and stain kit (Affymetrix)
  • Software: GeneChip Analysis Suite (Affymetrix) and Affymetrix Microarray Suite
  • Partek Genomics Suite (http://www.partek.com) or GeneSpring software (http://www.agilent.com)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Basic Protocol 3: Analysis of miRNA Expression by Two‐Step Quantitative RT‐PCR (qPCR)

  Materials
  • TRIzol reagent (Life Technologies)
  • Neural‐differentiated ESCs ( protocol 1) in 6‐well plates)
  • TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) for cDNA synthesis
  • TaqMan miNA assay containing: 5× stem loop RT‐primers for reverse‐transcription step and specific for miRNAs of interest and a 20× real‐time probe containing miRNA‐specific forward and reverse primers, as well as miRNA‐specific fluorescent MGB probe for real‐time PCR step (the TaqMan miRNA assay search tool is provided on the Molecular Probes Web page: http://www.lifetechnologies.com/us/en/home/life‐science/pcr/real‐time‐pcr/real‐time‐pcr‐assays.html)
  • TaqMan Fast Advanced PCR Master Mix for FAST PCR system or TaqMan Universal PCR Master Mix; no AmpErase UNG for machines operating in standard mode (optional)
  • 0.2‐ml RNase‐free PCR tubes
  • MicroAmp EnduraPlate Optical 96‐Well Fast GPLE Clear Reaction Plates with Barcode (Molecular Probes) for FAST machines or MicroAmp Optical 96‐well reaction plates for machine operating in standard mode
  • MicroAmp Clear Adhesive Films (Molecular Probes)
  • Thermal cycler: Applied Biosystems 7300 System, 7500 System, 7900 HT System, 7900 HT System, 7500 System, StepOnePlus, StepOnePlus, ViiA 7 System, StepOne, Mode, or StepOne: for FAST machines choose FAST or STANDARD mode dependent on the PCR master mix used
  • Additional reagents and equipment for handling RNA ( protocol 4)
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Figures

Videos

Literature Cited

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