Gene Expression Analysis of RNA Purified from Embryonic Stem Cells and Embryoid Body–Derived Cells Using a High‐Throughput Microarray Platform

Audrey Player1, Yonghong Wang2, Mahendra Rao3, Ernest Kawasaki1

1 National Cancer Institute, Bethesda, Maryland, 2 SAIC‐Frederick, Inc., NCI‐Frederick, Frederick, Maryland, 3 Johns Hopkins University School of Medicine, Baltimore, Maryland and Invitrogen Corp. Carlsbad California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1B.2
DOI:  10.1002/9780470151808.sc01b02s2
Online Posting Date:  September, 2007
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In this unit, starting with purified RNA, experimental protocols for performing microarrray expression analysis of embryonic stem cell lines compared to their corresponding differentiated embryoidal bodies are described. Methods for data analysis are suggested, with the goal of determining which genes are differentially expressed between the preparations. As an example, the use of the Affymetrix microarray expression platform is described, but alternative experimental options for analysis of RNA transcript levels are also summarized. This unit suggests quality control metrics, summazrizes the critical parameters necessary for obtaining reproducible experimental results, and outlines quantitative PCR methods for validating microarray results. Curr. Protoc. Stem Cell Biol. 2:1B.2.1‐1B.2.36. © 2007 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; embryoid bodies; RNA expression analysis; microarray platform

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Generating cDNA and In Vitro–Transcription (IVT) aRNA
  • Support Protocol 1: Assessment of ESC and EB RNA Quality Using the Agilent Bioanalyzer
  • Alternate Protocol 1: Two‐Cycle aRNA Amplification
  • Basic Protocol 2: Hybridizing, Washing, Staining, and Scanning of the Genechip
  • Basic Protocol 3: Data Analysis Using GCOS Batch Analysis Tool
  • Alternate Protocol 2: Data Analysis Using Modified PM‐Only Method
  • Basic Protocol 4: Real‐Time PCR Validation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Generating cDNA and In Vitro–Transcription (IVT) aRNA

  • ESC or EB RNA, quality assessed (see protocol 2)
  • Ambion MessageAmp II biotin‐enhanced kit (cat. no. 1791) containing:
    • T7 oligo(dT) primer
    • Arrayscript reverse transcription enzyme
    • RNAase inhibitor
    • 10× first‐strand buffer
    • dNTP mixture
    • 10× second‐strand buffer
    • DNA polymerase
    • RNAase H
    • T7 enzyme mixture
    • T7 10× reaction buffer
    • Biotin‐UTP mixture (which also includes unmodified NTPs)
    • Distilled water
  • Ambion MessageAmp II kit components (Optional; cat. no. 1751). Same as above except different in vitro transcription reagents, and random hexamer (day 2 reagent) as noted below:
    • T7 enzyme mixture
    • T7 10× reaction buffer
    • 75 mM T7 ATP solution
    • 75 mM T7 CTP solution
    • 75 mM T7 GTP solution
    • 75 mM T7 UTP solution
    • Random hexamer primer
    • Distilled water
    • Wash buffer (100% ethanol must be added before use)
    • cDNA binding buffer
    • aRNA binding buffer
    • aRNA filter cartridges
    • aRNA 1.7‐ml collection tubes
    • cDNA filter cartridges
    • cDNA 1.7‐ml collection tubes
    • 5× fragmentation buffer
  • 100% ethanol, ACS‐grade
  • 1 M Tris‐acetate, pH 8.1 (Trizma base, Sigma cat. no. T1503, pH adjusted with glacial acetic acid, Sigma cat. no. A628)
  • Magnesium acetate (Sigma cat. no. M2545)
  • Potassium acetate (Sigma cat. no. P5708)
  • DEPC‐treated water
  • Nuclease‐free 0.5‐ml tubes, non‐stick, sterile
  • Vortex
  • Microcentrifuge
  • Thermal cycler (with bonnet), incubator, or temperature block with adjustable temperatures
  • Vacuum centrifuge
  • Vacuum dryer
  • Spectrophotometer
  • Agilent Bioanalyzer or gel electrophoresis equipment, optional
  • 0.2‐µm vacuum filter unit

Support Protocol 1: Assessment of ESC and EB RNA Quality Using the Agilent Bioanalyzer

  • 1 µg RNA isolated from ∼106 ESC and EB cells
  • Agilent Nanochip, Lab‐on‐a chip (cat. no. 5067‐1511) including:
    • Detection chips
    • Cleaning chips
    • Syringe
    • Gel matrix
    • RNA ladder
    • Spin filters
    • Gel dye concentrate
    • NanoMarker
  • Nuclease‐free water
  • Low TE buffer (10 mM Tris⋅Cl, pH 7.4 and 1 mM EDTA)
  • RNaseZap (Ambion cat. no. 9780)
  • Agilent Bioanalyzer 2100 consisting of:
    • Priming station
    • Bioanalyzer vortexer
  • 70°C heating block
  • Centrifuge
  • 1.5‐ml nuclease‐free tubes

Alternate Protocol 1: Two‐Cycle aRNA Amplification

  • cDNA (see protocol 1, steps 1 to 24)
  • Ambion MessageAmp II (cat. no. 1751) or Ambion MessageAmp II biotin‐enhanced (cat. no. 1791)
  • Random hexamer

Basic Protocol 2: Hybridizing, Washing, Staining, and Scanning of the Genechip

  • Genechip eukaryotic hybridization control kit (Affymetrix cat. no. 900299; prokaryotic control samples used for eukaryotic samples)
  • Fragmented ES or EB aRNA (see protocol 1 or protocol 3)
  • Herring sperm DNA (Invitrogen Life Technologies cat. no. 1563‐4017)
  • 50 mg/ml acetylated bovine serum albumin (BSA; Invitrogen Life Technologies cat. no. 15561‐020)
  • 2× hybridization buffer (Quality Biological cat. no. 351‐289‐061 or see recipe)
  • Nonstringent wash buffer (Quality Biological cat. no. 351‐287‐131 or see recipe)
  • 2× MES stain buffer (Quality Biological cat. no. 351‐288‐101 or see recipe)
  • Streptavidin, R‐phycoerythrin streptavidin conjugate (SAPE; Invitrogen Life Technologies cat. no. S‐866)
  • Goat anti‐streptavidin antibody (Vector Laboratories, cat. no. BA‐0500), biotinylated
  • Goat IgG (Sigma‐Aldrich, cat. no. I 5256), reagent‐grade
  • Stringent wash buffer (Quality Biological cat. no. 351‐286‐131 or see recipe)
  • 45° and 95°C heating blocks
  • Affymetrix Genechip hybridization rotating oven
  • Affymetrix Fluidics 450 wash and stain station
  • 1.5‐ to 1.7‐ml nuclease‐free tubes
  • Affymetrix Genechip scanner 3000
  • Genechip operating system software (GCOS)

Basic Protocol 3: Data Analysis Using GCOS Batch Analysis Tool

  • Affymetrix GCOS microarray platform
  • GCOS software (available free of charge with the purchase of the Affymetrix microarray platform)

Alternate Protocol 2: Data Analysis Using Modified PM‐Only Method

  • First‐strand cDNA (see protocol 1, steps 1 to 9)
  • RNase H (10 U/µl; Ambion cat. no. AM12292)
  • Human glucoronidase (GUS) predeveloped control primers (Applied Biosystems cat. no. 4326320E)
  • Q‐PCR primers (Integrated DNA Technologies)
  • 2× AmpliGold PCR master mix (Applied Biosystems cat. no. E08415)
  • 1% (w/v) agarose gel, optional
  • SYBR Green PCR master mix (Applied Biosystems cat. no. 4309155)
  • 37°C water bath
  • Applied Biosystems 7900HT real‐time PCR system
  • BioAnalyzer DNA Lab‐on‐a‐chip (Agilent cat. no. 5067‐1504), optional
  • Optical adhesive covers (Applied Biosystems cat. no. 4311971)
  • Centrifuge (e.g., Eppendorf 5804)
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