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

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

1Johns Hopkins University School of Medicine, Baltimore, Maryland
2National Cancer Institute, Frederick, Maryland
3Dana‐Farber Cancer Institute, Boston, Massachusetts
4Brigham and Women's Hospital, Boston, Massachusetts
5Rutgers University, Piscataway, New Jersey





Publication Name: 
Current Protocols in Human Genetics
Unit Number: 
Unit 11.7
DOI: 
10.1002/0471142905.hg1107s53
Online Posting Date: 
April, 2007
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Abstract

This unit provides a protocol for performing serial analysis of gene expression (SAGE). 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.

Keywords: Genomics; mRNA; expression profiling; DNA sequencing

     
 
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Table of Contents

  • Unit 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
  • 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 (Life Technologies):
    • 5× first-strand buffer
    • DEPC-treated double-distilled water (DEPC ddH2O)
    • 1× first-strand buffer: dilute from 5× stock in DEPC ddH2O
    • 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 2D)
  • 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 Support Protocol 3):
  • 5 U/µl (high-concentration) T4 DNA ligase (Life Technologies)
  • 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 2D)
  • 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 2D)
  • 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 (Life Technologies)
  • 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 2D) containing 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 2D)
  • 1-kb DNA ladder
  • pZErO-1 plasmid (Invitrogen)
  • SphI and NEBuffer 2 (New England Biolabs)
  • TE buffer, pH 8.0 (appendix 2D)
  • SOC medium (appendix 2D)
  • 0.01 ng/µl pUC19 control DNA
  • DH10B Electromax competent cells, –70°C (Life Technologies)
  • LB medium (appendix 2D; optional)
  • LB plates with 100 µg/ml ampicillin (appendix 2D)
  • 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 2D)
  • 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 micro-electroporation cuvettes (BioRad)
  • Bio-Rad gene pulser electroporator or equivalent
  • 15-ml culture tubes
  • Additional reagents and equipment for determining integrity of cDNA by PCR (see Support Protocol 1), optimizing ditag PCR conditions (see Support Protocol 2), agarose gel electrophoresis (unit 2.7), ethanol precipitation (appendix 3C), polyacrylamide gel electrophoresis (cpmb unit 2.7) and direct sequencing of PCR products (cpmb 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

 Materials (also see Basic Protocol 1)
  • 350 ng/µl 5¢ and 3¢ primers (e.g., Integrated DNA Technology)
  • Bead suspension (see Basic Protocol 1, step 13)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

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

 Materials
  • SuperScript Choice System cDNA synthesis kit (Life Technologies):
    • DEPC ddH2O
    • 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 2D)
  • 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 Support Protocol 1)
  • 5 U/µl (high-concentration) T4 DNA ligase (Life Technologies)
  • 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 (Life Technologies)
  • 4% to 20% TBE acrylamide gel (Novex)
  • 1-kb ladder
  • 1× SYBR green I (Roche Diagnostics) in TBE buffer (appendix 2D)
  • 5 M betaine: prepare monohydrate salt (Sigma) in PCR-grade ddH2O
  • 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 (cpmb unit 4.2) agarose gel electrophoresis (unit 2.7) and sequencing (cpmb unit 7.4A)

Support Protocol 3: Phosphorylating and Annealing Linkers

 Additional Materials (also see Basic Protocol 1)
  • 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)
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 11.7.1
    The steps of a SAGE experiment.

    View Image
  • Figure 11.7.2
    Steps of an rSAGE experiment.

    View Image
  • Figure 11.7.3
    Screen shot of a sample SAGE data file. SAGE data file needs to be in tab-delimited format. All columns of SAGE libraries (tag counts) need to be arranged next to each other. Column 1 is SAGE tag, columns 2 to 6 are tag counts for 5 different SAGE libraries. For online version, the column headers are removed to keep data unidentifiable by other users.

    View Image
  • Figure 11.7.4
    Screen shot of the main page of the online version of the SAGE Data Analysis Application.

    View Image
  • Figure 11.7.5
    Screen shot for SAGE data significance analysis.

    View Image
  • Figure 11.7.6
    Selection of libraries 1, 3, and 5 for significance analysis.

    View Image
  • Figure 11.7.7
    Results from the significance analysis. Column 1 is the SAGE tag, columns 2 to 6 are 5 different SAGE libraries, column 7 is calculated P-value.

    View Image
  • Figure 11.7.8
    Annotated results after tag matching with SAGEmap. Column 1 is the SAGE tag, columns 2 to 6 are 5 different SAGE libraries, column 7 is calculated P-value, column 8 is organism (Hs = Homo sapiens), column 9 is unigene ID, column 10 is gene symbol and gene description.

    View Image
  • Figure 11.7.9
    Screen shot shows cluster #2 and all of its members after clicking on the graph of cluster #2.

    View Image

Literature Cited

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. Wiley, New York and London.
    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(10) 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...

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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