Serial Analysis of Gene Expression (SAGE): Experimental Method and Data Analysis

Seth Blackshaw1, Brad St. Croix2, Kornelia Polyak3, Jae Bum Kim4, Li Cai5

1 Johns Hopkins University School of Medicine, Baltimore, Maryland, 2 National Cancer Institute, Frederick, Maryland, 3 Dana‐Farber Cancer Institute, Boston, Massachusetts, 4 Brigham and Women's Hospital, Boston, Massachusetts, 5 Rutgers University, Piscataway, New Jersey
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25B.6
DOI:  10.1002/0471142727.mb25b06s80
Online Posting Date:  October, 2007
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Abstract

Serial analysis of gene expression (SAGE) involves the generation of short fragments of DNA, or tags, from a defined point in the sequence of all cDNAs in the sample analyzed. This short tag, because of its presence in a defined point in the sequence, is typically sufficient to uniquely identify every transcript in the sample. SAGE allows one to generate a comprehensive profile of gene expression in any sample desired from as little as 100,000 cells or 1 µg of total RNA. SAGE generates absolute, rather than relative, measurements of RNA abundance levels, and this fact allows an investigator to readily and reliably compare data to those produced by other laboratories, making the SAGE data set increasingly useful as more data is generated and shared. Software tools have also been specifically adapted for SAGE tags to allow cluster analysis of both public and user‐generated data. Curr. Protoc. Mol. Biol. 80:25B.6.1‐25B.6.39. © 2007 by John Wiley & Sons, Inc.

Keywords: Genomics; mRNA; expression profiling; DNA sequencing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: MicroSAGE
  • Support Protocol 1: Verifying cDNA Production by PCR Analysis
  • Support Protocol 2: Optimizing Ditag PCR Amplification
  • Basic Protocol 2: Reverse Cloning Unknown SAGE Tags (rSAGE)
  • Support Protocol 3: Phosphorylating and Annealing Linkers
  • Basic Protocol 3: Using the SAGE Data Analysis Application
  • Reagents and Solutions
  • Commentary
  • Appendix: Algorithm for Poisson‐Based Significance Analysis
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: MicroSAGE

  Materials
  • Dynabeads mRNA DIRECT kit (Dynal Biotech):
    • Dynabeads oligo(dT) 25
    • Lysis/binding buffer
    • Washing buffer A: add 1 µl 20 mg/ml molecular‐biology‐grade glycogen (Roche Diagnostics) per milliliter
    • Washing buffer B
  • Cells or tissue of interest
  • SuperScript Choice System cDNA synthesis kit (Invitrogen):
    • 5× first‐strand buffer
    • DEPC‐treated (unit 4.1) double‐distilled water (DEPC ddH 2O)
    • 1× first‐strand buffer: dilute from 5× stock in DEPC ddH 2O
    • 0.1 M DTT
    • 10 mM dNTP
    • 200 U/µl SuperScript II reverse transcriptase
    • 5× second‐strand buffer
    • 10 U/µl E. coli DNA ligase
    • 10 U/µl E. coli DNA polymerase I
    • 2 U/µl E. coli RNase H
    • 1× and 5× T4 DNA ligase buffer
    • 1 U/µl T4 DNA ligase
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 1× BW buffer (see recipe)/2× BSA (New England Biolabs)/0.1% (w/v) SDS
  • 1× BW buffer/2× BSA
  • 1× NEBuffer 4 (New England Biolabs)/2× BSA
  • LoTE buffer (see recipe)
  • 100× BSA (New England Biolabs)
  • 10 U/µl NlaIII and 10× NEBuffer 4 (New England Biolabs): store at −80°C
  • 1× BW buffer/2× BSA/1% (v/v) Tween 20
  • Annealed linkers (see protocol 5)
  • 5 U/µl (high‐concentration) T4 DNA ligase (Invitrogen)
  • 2 U/µl BsmFI (New England Biolabs)
  • PC8 (see recipe)
  • SeeDNA (Amersham Pharmacia Biotech)
  • 3:1 solution of 20 mg/ml glycogen/SeeDNA (optional)
  • 3 M sodium acetate ( appendix 22)
  • 70% and 100% ethanol
  • Klenow fragment of DNA polymerase I and 10× buffer (Amersham Pharmacia Biotech) or Roche Buffer H
  • 3 mM Tris·Cl, pH 7.5 ( appendix 22)
  • 10× SAGE PCR amplification buffer (see recipe)
  • DMSO (Sigma)
  • PCR primers (see recipe):
    • 350 ng/µl primers 1 and 2
    • 350 ng/µl M13 forward and reverse primers
  • 5 U/µl Platinum Taq DNA polymerase (Invitrogen)
  • 20 mg/ml glycogen (Roche Diagnostics)
  • 7.5 M ammonium acetate (Sigma)
  • Dry ice/methanol bath
  • 5× loading buffer: 50 mM EDTA/50 mM Tris·Cl, pH 8.0 ( appendix 22)/50% (v/v) glycerol
  • 20% (w/v) polyacrylamide/TBE minigels (Novex)
  • 20‐bp DNA ladder (GenSura)
  • 10,000× SYBR Green I (Roche Diagnostics)
  • 1× TBE ( appendix 22)
  • 1‐kb DNA ladder
  • pZErO‐1 plasmid (Invitrogen)
  • SphI and NEBuffer 2 (New England Biolabs)
  • TE buffer, pH 8.0 ( appendix 22)
  • SOC medium (unit 1.8)
  • 0.01 ng/µl pUC19 control DNA
  • DH10B Electromax competent cells, −70°C (Invitrogen)
  • LB medium (unit 1.1; optional)
  • LB plates with 100 µg/ml ampicillin (unit 1.1)
  • 10‐cm zeocin‐containing low‐salt LB plate (see recipe)
  • 10:1 U/µl Taq/Pfu polymerase (Stratagene)
  • Exonuclease I (USB)
  • Shrimp alkaline phosphatase (USB)
  • 50 mM Tris·Cl, pH 8.0 ( appendix 22)
  • 0.5‐, 1.5‐, 2.0‐ml RNase‐free No‐stick siliconized microcentrifuge tubes (Ambion)
  • Magnetic rack for 1.5‐ml microcentrifuge tubes (Dynal Biotech)
  • Tissue homogenizer (e.g., Polytron PT1200, Brinkmann Instruments)
  • 23‐G needles and 1‐ml syringes
  • 200‐µl aerosol‐barrier pipet tips
  • 16° and 65°C water baths, heat blocks, or equivalent
  • 96‐well PCR plates
  • 50‐ml conical tubes
  • Tabletop centrifuge with swinging‐bucket rotor
  • Gel‐loading tips
  • UV box and SYBR green or UV filter
  • 0.5‐ml microcentrifuge tubes with ∼0.5‐mm holes in the bottom: pierce from the inside out with a 21‐G needle
  • Spin‐X centrifuge‐tube filters (Costar)
  • Long‐wavelength UV source
  • 0.1‐mm disposable microelectroporation cuvettes (Bio‐Rad)
  • Gene Pulser electroporator (Bio‐Rad) or equivalent
  • 15‐ml culture tubes
  • Additional reagents and equipment for determining integrity of cDNA by PCR (see protocol 2), optimizing ditag PCR conditions (see protocol 3), agarose gel electrophoresis (unit 2.5), ethanol precipitation (unit 2.1), polyacrylamide gel electrophoresis (unit 2.7) and direct sequencing of PCR products (unit 15.2)
NOTE: Prepare Dynabeads, washing solutions, and 5× first‐strand mix before thawing and collecting cells.

Support Protocol 1: Verifying cDNA Production by PCR Analysis

  • 350 ng/µl 5′ and 3′ primers (e.g., Integrated DNA Technology)
  • Bead suspension (see protocol 1, step 13)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)

Support Protocol 2: Optimizing Ditag PCR Amplification

  Materials
  • SuperScript Choice System cDNA synthesis kit (Invitrogen):
    • DEPC ddH 2O
    • 5× first‐strand buffer
    • 0.1 M DTT
    • 10 mM dNTP
    • 200 U/µl SuperScript II reverse transcriptase
    • 5× second‐strand buffer
    • 10 U/µl E. coli DNA ligase
    • 10 U/µl E. coli DNA polymerase I
    • 2 U/µl E. coli RNase H
    • 5 U/µl T4 DNA polymerase
    • 1× and 5× T4 DNA ligase buffer
  • 1 µg/µl gel‐purified BRS1 primer (see recipe)
  • 0.5 M EDTA, pH 7.5 ( appendix 22)
  • PC8 (see recipe)
  • SeeDNA (Amersham Pharmacia Biotech)
  • 7.5 M ammonium acetate (Sigma)
  • 70% and 100% ethanol
  • LoTE buffer (see recipe)
  • 100× BSA (New England Biolabs)
  • 10 U/µl NlaIII and 10× NEBuffer 4 (New England Biolabs)
  • Streptavidin Dynabeads (Dynal)
  • 1× BW buffer (see recipe)
  • Annealed linkers (see protocol 2)
  • 5 U/µl (high‐concentration) T4 DNA ligase (Invitrogen)
  • 1× BW buffer/1× BSA
  • 1× NEBuffer 4/1× BSA
  • 100× BSA
  • 10 U/µl AscI (New England Biolabs)
  • 10× SAGE PCR buffer (see recipe)
  • DMSO
  • PCR primers (see recipe):
    • 350 ng/µl M13 forward primer
    • 350 ng/µl primer 2
  • 5 U/µl Platinum Taq DNA polymerase (Invitrogen)
  • 4% to 20% TBE acrylamide gel (Novex)
  • 1‐kb ladder
  • 1× SYBR green I (Roche Diagnostics) in TBE buffer ( appendix 22)
  • 5 M betaine: prepare monohydrate salt (Sigma) in PCR‐grade ddH 2O
  • SAGE tag–specific primer (see recipe)
  • Qiaquick gel‐extraction kit (Qiagen):
    • Qiaquick columns
    • EB Buffer
  • TOPO TA Cloning Kit with pCR2.1 vector (Invitrogen) or TOPO TA Cloning Kit for Sequencing with pCR4‐TOPO vector (Invitrogen)
  • 16°, 50°, and 70°C water baths, heat blocks, or equivalent
  • 1.5‐ml No‐stick siliconized microcentrifuge tubes (Ambion)
  • Magnetic rack for 1.5‐ml microcentrifuge tubes(Dynal)
  • 1.5‐ml nonsiliconized nuclease‐free microcentrifuge tubes
  • Additional reagents and equipment for preparing total RNA (unit 4.2), agarose gel electrophoresis (unit 2.5), and sequencing (unit 7.4)

Basic Protocol 2: Reverse Cloning Unknown SAGE Tags (rSAGE)

  • Linkers 1A, 1B, 2A, and 2B (see recipe)
  • 10× kinase buffer (New England Biolabs)
  • 10 mM ATP
  • 10 U/µl T4 polynucleotide kinase (New England Biolabs)
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Figures

Videos

Literature Cited

   Allinen, M., Beroukhim, R., Cai, L., Brennan, C., Lahti‐Domenici, J., Huang, H., Porter, D., Hu, M., Chin, L., Richardson, A., Schnitt, S., Sellers, W.R., and Polyak, K. 2004. Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell 6:17‐32.
   Angelastro, J.M., Kenzelmann, M., and Muhlemann, K. 1999. Substantially enhanced cloning efficiency of SAGE (serial analysis of gene expression) by adding a heating step to the original protocol. Nucl. Acids Res. 27:917‐918.
   Audic, S. and Claverie, J. M. 1997. The significance of digital gene expression profiles. Genome Res. 7:986‐995.
   Blackshaw, S., Harpavat, S., Trimarchi, J., Cai, L., Huang, H., Kuo, W.P., Weber, G., Lee, K., Fraioli, R.E., Cho, S.H., Yung, R., Asch, E., Wong, W.H., and Cepko, C.L. 2004. Genomic analysis of mouse retinal development. PLoS Biol. 2:E247.
   Boon, K., Osorio, E.C., Greenhut, S.F., Schaefer, C.F., Shoemaker, J., Polyak, K., Morin, P.J., Beutow, K.H., Strausberg, R.L., De Souza, S.J., Riggins, G.J. 2002. An anatomy of normal and malignant gene expression. Proc. Natl. Acad. Sci. U.S.A. 99:11287‐11292.
   Cai, L., Huang, H., Blackshaw, S., Liu, J.S., Cepko, C., and Wong, W.H. 2004. Clustering analysis of SAGE data using a Poisson approach. Genome Biol. 5(7) R51.
   Datson, N.A., van der Perk‐de Jong, J., van den Berg, M.P., de Kloet, E.R., and Vreugdenhil, E. 1999. MicroSAGE: A modified procedure for serial analysis of gene expression in limited amounts of tissue. Nucl. Acids Res. 27:1300‐1307.
   Gorski, S.M., Chittaranjan, S., Pleasance, E.D., Freedman, J.D., Anderson, C.L., Varhol, R.J., Coughlin, S.M., Zuyderduyn, S.D., Jones, S.J., and Marra, M.A. 2003. A SAGE approach to discovery of genes involved in autophagic cell death. Curr. Biol. 13:358‐363.
   Halascheck‐Wiener, J., Khattra, J.S., McKay, S., Pouzyrev, A., Stott, J.M., Yang, G.S., Holt, R.A., Jones, S.J., Marra, M.A., Brooks‐Wilson, A.R., and Riddle, D.L. 2005. Analysis of long‐lived C. elegans daf‐2 mutants using serial analysis of gene expression. Genome Res. 15:603‐615.
   Hartigan, J. 1975. Clustering Algorithms. John Wiley & Sons, New York.
   Hosack, D.A., Dennis, G., Jr., Sherman, B.T., Lane, H.C., and Lempicki, R.A. 2003. Identifying biological themes within lists of genes with EASE. Genome Biol. 4:R70.
   Klimaschewski, L., Tang, S., Vitolo, O.V., Weissman, T.A., Donlin, L.T., Shelanski, M.L., and Greene, L.A. 2000. Identification of diverse nerve growth factor‐regulated genes by serial analysis of gene expression (SAGE) profiling. Proc. Natl. Acad. Sci. U.S.A. 97:10424‐10429.
   Lepourcelet, M., Tou, L., Cai, L., Sawada, J., Lazar, A.J., Glickman, J.N., Williamson, J.A., Everett, A.D., Redston, M., Fox, E.A., Nakatani, Y., and Shivdasani, R.A. 2005. Insights into developmental mechanisms and cancers in the mammalian intestine derived from serial analysis of gene expression and study of the hepatoma‐derived growth factor (HDGF). Development 132:415‐427.
   Neilson, L., Andalibi, A., Kang, D., Coutifaris, C., Strauss, J.F. 3rd, Stanton, J.A., and Green, D.P. 2000. Molecular phenotype of the human oocyte by PCR‐SAGE. Genomics 63:13‐24.
   Polyak, K., Xia, Y., Zweier, J.L., Kinzler, K., and Vogelstein, B. 1997. A model for p53 induced apoptosis. Nature 389:300‐305.
   St. Croix, B., Rago, C., Velculescu, V., Traverso, G., Romans, K.E., Montgomery, E., Lal, A., Riggins, G.J., Lengauer, C., Vogelstein, B., and Kinzler, K.W. 2000. Genes expressed in human tumor endothelium. Science 289:1197‐1202.
   Velculescu, V.E., Zhang, L., Vogelstein, B., and Kinzler, K.W. 1995. Serial analysis of gene expression. Science 270:484‐487.
   Velculescu, V.E., Zhang, L., Zhou, W., Vogelstein, J., Basrai, M.A., Bassett, D.E., Hieter, P., Vogelstein, B., and Kinzler, K.W. 1997. Characterization of the yeast transcriptome. Cell 88:243‐251.
   Virlon, B., Cheval, L., Buhler, J.M., Billon, E., Doucet, A., and Elalouf, J.M. 1999. Serial microanalysis of renal transcriptomes. Proc. Natl. Acad. Sci. U.S.A. 96:15286‐15291.
   Zhang, L., Zhou, W., Velculescu, V.E., Kern, S.E., Hruban, R.H., Hamilton, S.R., Vogelstein, B., and Kinzler, K.W. 1997. Gene expression profiles in normal and cancer cells. Science 276:1268‐1272.
Internet Resources
  http://www.sagenet.org
  SAGEnet. Contains instructions for obtaining SAGE analysis software, downloadable SAGE libraries from human, mouse and yeast, and a comprehensive bibliography of SAGE papers.
  http://www.ncbi.nlm.nih.gov/SAGE
  Serial analysis of gene expression at NCBI.
  http://www.ncbi.nlm.nih.gov/CGAP
  Cancer Genome Anatomy project. Contains full downloadable predicted tag data for human, mouse, rat, zebrafish, and cow. Also contains a large number of downloadable human SAGE libraries (containing >3.5 million total tags), as well as tools for submitting SAGE data for public access and tools for searching tag abundance levels in the publicly available human SAGE data.
  http://www.umich.edu/∼ehm/eSAGE
  eSAGE at University of Michigan. Helpful software for SAGE data analysis.
  http://www.invitrogen.com
  iSAGE at Invitrogen. Integrated kit and software package for conducting microSAGE. The protocol used is very similar to the one described here.
  http://arep.med.harvard.edu/labgc/adnan/projects/Utilities/mergesagetags.html
  Merge SAGE tags at Harvard Medical School. Helpful tool for merging SAGE data files and downloaded predicted tag identify files (from NCBI).
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