Preparation of mRNA for Expression Monitoring

Michael C. Byrne1, Maryann Z. Whitley1, Maximillian T. Follettie1

1 Genetics Institute/Wyeth Research, Cambridge, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.26
DOI:  10.1002/0471142301.ns0426s16
Online Posting Date:  November, 2001
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Abstract

The ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A+) versus total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.

     
 
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Table of Contents

  • Unit Introduction
  • Strategic Planning
  • Basic Protocol: Amplification of mRNA for Expression Monitoring and Hybridization to Oligonucleotide Array Chips
  • Support Protocol 1: In Vitro Transcription of Control Genes and Preparation of Transcript Pools
  • Alternate Protocol: Solid‐Phase Reversible Immobilization (SPRI) Purification of cDNA and IVT Products
  • Support Protocol 2: Quantitation of cDNA
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Amplification of mRNA for Expression Monitoring and Hybridization to Oligonucleotide Array Chips

 Materials
  • SuperScript cDNA kit (Life Technologies), including:
  •  5× First Strand Buffer
  •  200 U/µl SuperScript II reverse transcriptase
  •  5× Second Strand Buffer
  •  10 mM dNTPs
  •  10 U/µl E. coli ligase
  •  2 U/µl E. coli RNase H
  •  10 U/µl E. coli DNA polymerase
  •  5 U/µl T4 DNA polymerase
  • T7T24 primer: 5′‐GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG TTT TTT TTT TTT TTT TTT TTT TTT‐3′ (custom synthesis; HPLC purification is recommended)
  • RNase inhibitor (Life Technologies or Ambion)
  • RNase‐free H2O (see appendix 2A for DEPC treatment of solutions; prepare from glass‐distilled H2O)
  • Sample RNA: poly(A)+ or total RNA
  • Sense control transcript pool (see Support Protocol 1)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (molecular biology grade; appendix 2A)
  • 7.5 M ammonium acetate
  • Absolute ethanol
  • 70% (v/v) ethanol in RNase‐free H2O, prechilled to −20°C
  • 10× transcription buffer (Ambion)
  • 10× rNTP mix (see recipe)
  • 100 mM dithiothreitol (DTT)
  • 10 mM Bio‐11‐CTP and Bio‐11‐UTP (Enzo Diagnostics)
  • 2500 U/µl T7 RNA polymerase (Epicentre)
  • RNeasy mini columns with RLT and RPE buffers and collection tubes (Qiagen)
  • 5× fragmentation buffer (see recipe)
  • 20× SSPE (Bio‐Whittaker; also see appendix 2A)
  • 0.5% (v/v) Triton X‐100 (molecular biology grade; Sigma) in RNase‐free H2O
  • 10 mg/ml herring sperm DNA (Promega)
  • 500 pM Bio948 (see recipe)
  • 20× antisense control transcript pool (see Support Protocol 1)
  • 6× SSPET: 6× SSPE containing 0.005% (v/v) Triton X‐100
  • Thermal cycler (e.g., Perkin‐Elmer 9600 PCR machine with heated lid)
  • 0.1‐ to 10‐µl filtered micropipet tips (Continental)
  • Lyophilizer
  • Small, thin‐walled PCR tubes
  • GeneChip (Affymetrix)
  • 1‐ to 200‐µl filtered gel‐loading micropipet tips (Fisher)
  • Rotisserie‐type rotator (Appropriate Technical Resources)
  • 50°C oven
  • Additional reagents and equipment for quantitation of cDNA (see Support Protocol 2), for quantitation of DNA by spectrophotometry (appendix 1K), and for washing, staining, and scanning the GeneChip (see manufacturer's instructions)

NOTE: Many buffers and enzymes are supplied with the SuperScript II cDNA kit. Kit enzymes that are limiting may be ordered separately from Life Technologies.

NOTE: All temperature‐controlled reactions are performed in an appropriate thermal cycler.

Support Protocol 1: In Vitro Transcription of Control Genes and Preparation of Transcript Pools

 Additional Materials (also see Basic Protocol)
  • Plasmids (Table 4.26.1; ATCC #87482 to 87490)
  • 2500 U/µl T3 RNA polymerase (Enzo Diagnostics)
  • 25 mM 4rNTP mix: 25 mM each rGTP, rCTP, rATP, and UTP (Ultrapure; Pharmacia Biotech) in RNase‐free H2O
  • Additional reagents and equipment for purifying IVT products (see Alternate Protocol)
     
    Table 4.26.1 Preparation of Plasmid Template Controls
    NameaATCC #Transcript size (kb)Sense RNAAntisense RNA
    Linearize withPolymerize withLinearize withPolymerize with
    pGIBS‐LYS b874821.0NotIT3
    pGIBS‐PHE b874831.3NotIT3
    pGIBS‐THR b874842.0NotIT3
    pGIBS‐TRP b874852.5NotIT3  
    pGIKS‐BioB874871.1XhoIT7
    pGIKS‐BioC874880.8XhoIT7
    pGIKS‐BioD874890.7XhoIT7
    pGIKS‐CRE874901.0XhoIT7
     a Abbreviations: BioB, BioC, and BioD are cloned fragments from the E. coli bioB, bioC, and bioD genes, respectively. LYS, PHE, THR, and TRP are fragments from the Bacillussubtilis lysA, pheA, thrBC, and trpEDCF genes, respectively. CRE is a fragment from the Cre recombinase derived from E. coli bacteriophage P1. PGIBS and pGIKS are derived from the Bluescript KS II vector (Stratagene).
     b pGIBS‐LYS, −PHE, −THR, −TRP, and DAP contain a 40‐nucleotide synthetic poly(A) tract at the 3′ end of the respective genomic fragments derived from B. subtilis. Sense IVT transcripts derived from NotI‐linearized plasmid templates will contain the artificial poly(A) tail. Plasmids linearized with BamHI prior to T3 IVT will generate sense transcripts without the synthetic poly(A) tract.

Alternate Protocol: Solid‐Phase Reversible Immobilization (SPRI) Purification of cDNA and IVT Products

 Materials
  • Carboxy‐coated magnetic beads (PerSeptive BioSystems for cDNA purification; Bangs Laboratories for IVT purification)
  • 0.5 M EDTA (appendix 2A)
  • Sample to be purified: cDNA (see Basic Protocol, step ) or IVT RNA (see Basic Protocol, step , or see Support Protocol 1, step )
  • 2.5 M NaCl/20% (w/v) PEG 8000 (molecular biology grade; RNase free)
  • 70% (v/v) ethanol in RNase‐free H2O
  • 10 mM Tris acetate, pH 7.8 (RNase free)
  • Magnetic stand (CPG)
  • Additional reagents and equipment for determining concentration of cDNA (see Support Protocol 2) or RNA (appendix 1K)

Support Protocol 2: Quantitation of cDNA

 Materials
  • PicoGreen dsDNA Quantitation Kit (Molecular Probes), including:
  •  100 ng/µl standard DNA stock solution
  •  20× TE buffer
  •  PicoGreen reagent
  • cDNA to be quantitated (see Basic Protocol and Alternate Protocol)
  • Black‐walled 96‐well plate (Corning)
  • Fluorimager (Molecular Dynamics, model FSI)
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Figures

  •  FigureFigure 4.26.1 Chip analysis overview. Abbreviations: GIT, guanidine isothiocyanate, PE, phycoerythrin; SPRI, solid‐phase reversible immobilization.
    View Image

Videos

Literature Cited

 Literature Cited
    DeAngelis, M.M., Wang, D.G., and Hawkins, T.L. 1995. Solid‐phase reversible immobilization for the isolation of PCR products. Nucl. Acids Res. 23:4742‐4743.
    Lockhart, D.J., Dong, H., Byrne, M.C., Follettie, M.T., Gallo, M.V., Chee, M.S., Mittmann, M., Wang, C., Kobayashi, M., Horton, H., and Brown, E.L. 1996. Expression monitoring by hybridization to high‐density oligonucleotide arrays. Nature Biotechnol. 14:1675‐1680.
    Schena, M., Shalon, D., Davis, R.W., and Brown, P.O. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467‐470.
 Key References
    Lockhart et al., 1996. See above.

This paper established that, in addition to its role as a resequencing tool, the oligonucleotide array could be used as a powerful methodology for transcriptional profiling.

    Schena et al., 1995. See above.

A seminal paper on the potential of robotics and microfabrication to accelerate cDNA analysis using a highly parallel format.

    Wodicka, L., Dong, H., Mittmann, M., Ho, M.H., and Lockhart, D.J. 1997. Genome‐wide expression monitoring in Saccharomyces cerevisiae. Nature Biotechnol. 15:1359‐1367.

Demonstration that oligonucleotide arrays can be used to simultaneously monitor expression of all genes of a eukaryotic organism.

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