Preparation of mRNA for Expression Monitoring

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

1 Genetics Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 22.2
DOI:  10.1002/0471142727.mb2202s49
Online Posting Date:  May, 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+) vs. 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

  • Strategic Planning
  • Basic Protocol 1: 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 1: Solid‐Phase Reversible Immobilization (SPRI) Purification of cDNA and IVT Products
  • Support Protocol 2: Quantitation of cDNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: 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
  • RNase inhibitor (Life Technologies or Ambion)
  • RNase‐free H 2O (see unit 4.1 for DEPC treatment of solutions; prepare from glass‐distilled H 2O)
  • Sample RNA: poly(A)+ or total RNA
  • Sense control transcript pool (see protocol 2)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (molecular biology grade; unit 2.1)
  • 7.5 M ammonium acetate ( appendix 22)
  • Absolute ethanol
  • 70% (v/v) ethanol in RNase‐free H 2O, prechilled to −20°C
  • 10× transcription buffer (Ambion)
  • recipe10× 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)
  • recipe5× fragmentation buffer (see recipe)
  • 20× SSPE (Bio‐Whittaker)
  • 0.5% (v/v) Triton X‐100 (molecular biology grade; Sigma) in RNase‐free H 2O
  • 10 mg/ml herring sperm DNA (Promega)
  • recipe500 pM Bio948 (see recipe)
  • 20× antisense control transcript pool (see protocol 2)
  • 6× SSPET: 6× SSPE containing 0.005% (v/v) Triton X‐100
  • Thermocycler (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 protocol 4), for quantitation of DNA by spectrophotometry ( appendix 3D), 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 thermocycler.

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

  • Plasmids (Table 22.2.1; 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 H 2O
  • Additional reagents and equipment for purifying IVT products (see protocol 3)
    Table 2.2.1   Additional Materials (also see protocol 1)   Additional MaterialsPreparation of Plasmid Template Controls

    Name a ATCC # Transcript size (kb) Sense RNA Antisense RNA
    Linearize with Polymerize with Linearize with Polymerize with
    pGIBS‐LYS b 87482 1.0 NotI T3
    pGIBS‐PHE b 87483 1.3 NotI T3
    pGIBS‐THR b 87484 2.0 NotI T3
    pGIBS‐TRP b 87485 2.5 NotI T3
    pGIKS‐BioB 87487 1.1 XhoI T7
    pGIKS‐BioC 87488 0.8 XhoI T7
    pGIKS‐BioD 87489 0.7 XhoI T7
    pGIKS‐CRE 87490 1.0 XhoI T7

     aAbbreviations: 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 Bacillus subtilis 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).
     bpGIBS‐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 1: 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 22)
  • Sample to be purified: cDNA (see protocol 1, step ) or IVT RNA (see protocol 1, step , or see protocol 2, step )
  • 2.5 M NaCl/20% (w/v) PEG 8000 (molecular biology grade; RNase free)
  • 70% (v/v) ethanol in RNase‐free H 2O
  • 10 mM Tris acetate, pH 7.8 (RNase free)
  • Magnetic stand (CPG)
  • Additional reagents and equipment for determining concentration of cDNA (see protocol 4) or RNA ( appendix 3D)

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 protocol 1 and protocol 3)
  • Black‐walled 96‐well plate (Corning)
  • Fluorimager (Molecular Dynamics, model FSI)
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Figures

Videos

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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