Gene Expression Analysis of Neural Cells and Tissues Using DNA Microarrays

Stanislav L. Karsten1, Lili C. Kudo1, Daniel H. Geschwind1

1 David Geffen School of Medicine at UCLA, Los Angeles, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.28
DOI:  10.1002/0471142301.ns0428s45
Online Posting Date:  October, 2008
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DNA microarrays pose specific challenges to those studying the central and peripheral nervous systems. Probably the most important involve difficulty in obtaining appropriate tissue for study, as well as the problems posed by cellular heterogeneity. This unit describes advances in the available technologies and provides protocols for cDNA microarray hybridization, including the use of PCR amplicons. Protocols are also provided for the two major methods for limiting cellular heterogeneity by study of RNA from single cell populations in high‐throughput microarray studies, laser capture microdissection (LCM), and automated fluorescent cell sorting (FACS‐array). Curr. Protoc. Neurosci. 45:4.28.1‐4.28.38. © 2008 by John Wiley & Sons, Inc.

Keywords: gene expression; flow sorting; single cell analysis; laser capture; microarray analysis

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Direct Labeling of cDNA using Klenow Fragment
  • Alternate Protocol 1: Direct Labeling using Reverse Transcriptase
  • Basic Protocol 2: Indirect Labeling and Detection of cDNA using PCR Amplification
  • Basic Protocol 3: Modified T7‐Based Two‐Round Amplification for Small Quantities of RNA Derived Through Laser Capture Microdissection (LCM) or Fluorescence‐Assisted Cell Sorting (FACS)
  • Support Protocol 1: Laser Capture Microdissection (LCM)
  • Support Protocol 2: Enzymatic Dissociation and Fluorescence‐Assisted Cell Sorting (FACS) of the Genetically Labeled Neurons from BAC‐EGFP Transgenic Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Direct Labeling of cDNA using Klenow Fragment

  • Sample: 0.8 µg/ml poly(A)+ RNA or 20 µg/ml total RNA (units 4.26& 5.3) in TE buffer ( 2.NaN)
  • SuperScript II cDNA kit (Invitrogen Life Technologies) containing:
    • 0.5 µg/µl oligo(dT) 12‐18
    • 5× first‐strand buffer
    • 0.1 M dithiothreitol (DTT)
    • 10 mM 4dNTP mix (10 mM each dATP, dCTP, dGTP, and dTTP)
    • 200 U/µl SuperScript II reverse transcriptase
    • 5× second‐strand buffer
    • 10 U/µl E. coli DNA polymerase I
    • 2 U/µl RNase H
    • 10 U/µl E. coli DNA ligase
    • 5× random primers (hexamers)
    • DEPC‐treated H 2O
  • 10 U/µl RNasin (Promega)
  • 15 mM β‐nicotinamide adenine dinucleotide (β‐NAD; Sigma)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 2A), prepared with molecular‐biology‐grade ingredients
  • Chloroform, molecular biology grade
  • 10 M ammonium acetate
  • 1 µg/µl glycogen carrier (Boehringer Mannheim)
  • 100% ethanol (molecular‐biology grade; Sigma), −20°C
  • 70% (v/v) ethanol diluted with DEPC‐treated H 2O, ice cold
  • 5× Klenow buffer (Stratagene)
  • 0.25 mM dCTP
  • 2.5 mM 3dNTP mix (2.5 mM each dATP, dGTP, and dTTP)
  • 1 mM cyanine‐3‐ and cyanine‐5‐dCTP (Cy3‐ and Cy5‐dCTP; GE Healthcare)
  • 5 U/µl Klenow fragment (exo) (Stratagene)
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • Hybridization buffer (see recipe)
  • 2× SSC ( appendix 2A)
  • 0.2× and 2× SSC/0.1% SDS
  • Microarray (commercial or custom made; see and Internet Resources)
  • Additional reagents and equipment for cDNA labeling and detection (see ) and measuring DNA concentration by absorption spectroscopy ( appendix 1K)
NOTE: Unless otherwise mentioned, all reagents are available from Invitrogen.

Alternate Protocol 1: Direct Labeling using Reverse Transcriptase

  • 100 mM 3dNTP mix (100 mM each dATP, dGTP, and dTTP; store up to several months at −20°C)
  • 100 mM dCTP
  • 0.5 µg/µl oligo(dT) 18‐20
  • 1 mM cyanine‐3‐ and cyanine‐5‐dCTP (Cy3‐dCTP and Cy5‐dCTP; Amersham Pharmacia Biotech)
  • 20 mM EDTA, pH 8.0 ( appendix 2A)
  • 0.5 M NaOH
  • 0.5 M HCl
  • 100% isopropanol

Basic Protocol 2: Indirect Labeling and Detection of cDNA using PCR Amplification

  • 10 U/µl DpnII and 10× buffer (NEB)
  • 1% and 1.2% agarose gels ( appendix 1N)
  • 1× TAE electrophoresis buffer ( appendix 1N)
  • T4 DNA ligase and 10× buffer (NEB)
  • 10 mM ATP
  • 2 µg/µl custom‐synthesized oligonucleotide adaptors R1 and R2 ( appendix 1A):
  • 10× PCR reaction buffer (Qiagen)
  • 25 mM MgCl 2 (Qiagen)
  • 10 mM 4dNTP mix (10 mM each dATP, dCTP, dGTP, and dTTP)
  • 5 U/µl Taq DNA polymerase (Qiagen) or AmpliTaq DNA polymerase (Perkin‐Elmer)
  • Small thin‐walled PCR tubes
  • Thermal cycler (e.g., Perkin‐Elmer 9600 PCR machine with heated lid)
  • Additional reagents and equipment for cDNA labeling and detection (see ), synthesis of double‐stranded cDNA ( protocol 1), restriction endonuclease digestion ( appendix 1M), agarose gel electrophoresis ( appendix 1N), and measuring DNA concentration by absorption spectroscopy ( appendix 1K)

Basic Protocol 3: Modified T7‐Based Two‐Round Amplification for Small Quantities of RNA Derived Through Laser Capture Microdissection (LCM) or Fluorescence‐Assisted Cell Sorting (FACS)

  • 2‐mercaptoethanol (2‐ME)
  • RNeasy Mini Kit (Qiagen) including:
    • Buffer RLT
    • Buffer RPE (concentrate)
    • RNase‐free H 2O
    • Carrier RNA
    • RNeasy MinElute Spin Columns in 2‐ml collection tubes
    • Buffer RW1
  • 96% to 100% ethanol
  • Total RNA sample (e.g., from protocol 5 or protocol 62)
  • Molecular‐biology‐grade H 2O (RNase‐, DNase‐, and protease‐free)
  • Low RNA Input Linear Amplification Kit PLUS Two‐Color (Agilent) including:
    • T7 promoter primer
    • 5× first strand buffer
    • 0.1 M DTT
    • 10 mM dNTP mix
    • MMLV Reverse Transcriptase (RT)
    • RNase Inhibitor
    • 4× transcription buffer
    • NTP mix
    • Inorganic pyrophosphatase
    • T7 RNA polymerase
    • 50% polyethylene glycol (PEG)
    • Cyanine 3‐CTP
    • Cyanine 5‐CTP
  • RNeasy Mini spin columns
  • Hybridization kit (Agilent) including:
    • 10× control targets/blocking agent
    • 25× fragmentation buffer
    • 2× hybridization buffer
    • Wash solution 1
    • Wash solution 2
    • Stabilization and drying solution
  • ND‐1000 spectrophotometer (NanoDrop Technologies) or Agilent Bioanalyzer 2100
  • Microarray slides (e.g., Agilent)
  • Additional reagents and equipment for cDNA labeling and detection (see )

Support Protocol 1: Laser Capture Microdissection (LCM)

  • Slide‐mounted tissue sections of interest (e.g., unit 1.1)
  • 75% and 95% ethanol prepared using molecular‐biology‐grade (RNase‐, DNase‐, and protease‐free) H 2O
  • 0.5% cresyl violet solution (see recipe)
  • 100% ethanol (molecular‐biology‐grade)
  • Xylene (molecular‐biology‐grade)
  • Coplin jars or staining dishes
  • PixCell IIe LCM System (Arcturus, Molecular Probes) or newer model

Support Protocol 2: Enzymatic Dissociation and Fluorescence‐Assisted Cell Sorting (FACS) of the Genetically Labeled Neurons from BAC‐EGFP Transgenic Mice

  • Freshly removed laboratory animal brain (e.g., from BAC‐EGFP transgenic mice)
  • Papain Dissociation System Kit (Worthington) including:
    • Vial 1: Earle's Balanced Salt Solution (EBSS)
    • Vial 2: papain‐containing L‐cysteine and EDTA
    • Vial 3: Deoxyribonuclease I (DNase)
    • Vial 4: Albumin ovomucoid inhibitor (AOI)
  • FACS buffer (see recipe)
  • 1 mg/ml propidium iodide (PI)
  • Rocking platform (optional)
  • Three manually pulled glass pipets of decreasing (0.1‐ to 0.2‐mm) tip diameter
  • Refrigerated centrifuge and sterile centrifuge tubes
  • FACS tubes
  • FACS instrument (e.g., FACSVantage SE Cell Sorter; BD Biosciences)
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Key References
   DeRisi et al., 1997. See above.
  Tour de force analysis of yeast gene expression done in the laboratory that started it all.
   Geschwind et al., 2001. See above.
  This paper demonstrates the power of using custom microarrays derived from RDA‐subtracted libraries for gene discovery and gene expression analysis in the central nervous system. It also emphasizes the utility of coupling microarray studies with methods such as in situ hybridization that provide high spatial resolution.
   Arlotta et al., 2005. See above.
  These two papers provide a first successful demonstration of automated cell sorting for collection of the genetically labeled neuronal subtypes and their subsequent analysis using high‐density oligonucleotide microarrays during development (Arlotta et al., ) and in adult tissue (Lobo et al., ). Detailed protocols are described and the specificity of microarray data is confirmed functionally from both young and adult animals.
   Lobo et al., 2006. See above.
  In this paper, laser capture microdissection (LCM) is coupled with microarray experiments to study gene expression in single neuronal types for the first time.
   Luo et al., 1999. See above.
  In this seminal paper, microarray analysis of human post‐mortem tissues is aimed to advance understanding of a complex neuropsychiatric disorder, schizophrenia.
   Mirnics et al., 2000. See above.
  Demonstration of the use of peripheral lymphoblasts from patients to identify changes in a complex disease of the brain (autism). This study showed that this approach could be used to identify expression changes that were relevant to the nervous system by validating changes in vivo in mice and in vitro in a neuronal cell line.
   Nishimura et al., 2007. See above.
  First demonstration of the power of network methods (systems biology) to identify key drivers of disease‐relevant biological alterations. Here, new therapeutic targets were identified in brain tumors.
   Horvath et al., 2006. See above.
  Seminal use of microarray technology for expression profiling used to classify CNS tumors and define prognosis.
   Pomeroy et al., 2002. See above.
  This important study demonstrates importance of strain background on gene expression in mice.
   Sandberg et al., 2000. See above.
  This seminal paper is the first demonstration of cDNA microarray analysis for high‐throughput gene expression studies.
   Schena et al., 1995. See above.
  The results presented demonstrate the reproducibility and reliability of cDNA microarray technology.
   Yue et al., 2001. See above.
Internet Resources
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