SAGE Analysis from 1 µg of Total RNA

Jerry Cai1, David Ash1, Ethylin Wang Jabs1

1 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.4
DOI:  10.1002/0471143030.cb1904s16
Online Posting Date:  November, 2002
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Abstract

Serial analysis of gene expression (SAGE) is a powerful transcription‐profiling method that allows simultaneous expression analysis of thousands of transcripts, provides absolute digital readout of the expression level, and identifies new genes. A disadvantage of SAGE is the relatively high amount of input RNA required. Consequently, several techniques have been developed to overcome this limitation, so that SAGE can be applied to very limited amounts of starting material, e.g., small biological samples such as tissue biopsies or microdissected materials. Here we describe a modified version of the original microSAGE protocol, which requires only 1 to 2 ug total RNA. This method avoids PCR amplification of cDNA and reamplification of ditags, procedures that potentially compromise the quantitative nature of this technique.

     
 
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Table of Contents

  • Basic Protocol 1: Construction of SAGE Library from 1 µg of Total RNA
  • Support Protocol 1: PCI Extraction
  • Alternate Protocol 1: Substituting Hsp92II for NlaIII as the Anchoring Enzyme
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Construction of SAGE Library from 1 µg of Total RNA

  Materials
  • 1 to 2 µg of total RNA from tissues or cells
  • DEPC‐treated H 2O ( appendix 2A)
  • Dynabeads mRNA Purification Kit (Dynal) including:
  •  Oligo(dT) 25 beads
  •  Binding buffer
  •  Washing buffer B
  • 1× first‐strand/glycogen buffer (see recipe)
  • Superscript Choice System for cDNA Synthesis kit (Invitrogen), including:
  •  5× first‐strand reaction buffer
  •  0.1 M dithiothreitol (DTT)
  •  10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP)
  •  SuperScript II reverse transcriptase
  •  40 U/µl RNaseOUT recombinant ribonuclease inhibitor
  •  5× second‐strand reaction buffer
  •  10 U/µl E. coli DNA polymerase I
  •  10 U/µl E. coli DNA ligase
  •  2 U/µl E. coli RNase H
  •  1 U/µl T4 DNA polymerase
  • 0.5 M EDTA, pH 7.5 ( appendix 2A)
  • 1× B&W/SDS/glycogen buffer (see recipe)
  • 1× B&W/BSA buffer (see recipe)
  • 1× NEBuffer 4/BSA solution (see recipe)
  • LoTE buffer (unit 19.3)
  • 100× (10 µg/µl) BSA (New England Biolabs; supplied with corresponding restriction enzymes)
  • 10× NEBuffer 4 (New England Biolabs; supplied with corresponding restriction enzymes)
  • 10 U/µl restriction endonuclease NlaIII (New England Biolabs)
  • 5 U/µl T4 DNA ligase and 10× ligase buffer (Invitrogen)
  • Annealed linker 1 and annealed linker 2 (see unit 19.3, protocol 6)
  • 7.5 M ammonium acetate
  • 20 µg/µl glycogen
  • 70% and 100% ethanol
  • 10 U/µl Klenow fragment of DNA polymerase (Amersham Pharmacia Biotech or USB)
  • Platinum Taq DNA polymerase (Invitrogen) and 10× PCR buffer
  • 16°, 50°, 65°, and 75°C water baths or heating blocks
  • Safe‐Lock tubes (Eppendorf) or microcentrifuge tubes
  • Magnetic Particle Collector (MPC; Dynal, cat. no. 120.20)
  • Sample mixer (Dynal, cat. no. 947.01)
  • 8‐well PCR strips, sterile (USA Scientific)
  • Thermal cycler
  • Additional reagents and equipment for PCR ( appendix 3F) and constructing a SAGE library (see unit 19.3, protocol 4)

Support Protocol 1: PCI Extraction

  Materials
  • DNA solution to be extracted
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol solution (PCI; Invitrogen)
  • Phase‐Lock Gel Heavy in 2‐ml tubes (Eppendorf)

Alternate Protocol 1: Substituting Hsp92II for NlaIII as the Anchoring Enzyme

  • 10 U/µl restriction endonuclease Hsp92II (Promega)
  • 100× BSA and 10× buffer K (provided with Hsp92II)
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Figures

Videos

Literature Cited

Literature Cited
  Datson, N.A., van der Perk‐de Jong, J., van den Berg, M.P., de Kloet, E.R., and Vregdenhil, E. 1999. MicroSAGE: A modified procedure for serial analysis of gene expression in limited amounts of tissue. Nucleic Acid Res. 27:1300‐1307.
  Neilson, L., Andalibi, A., Kang, D., Coutifaris, C., Strauss, J.F. 3rd, Santon, J.A., and Green, D.P. 2000. Molecular phenotype of the human oocyte by PCR‐SAGE. Genomics 63:13‐24.
  Peters, D.G., Kassam, A.B., Yonas, H., O'Hare, E.H., Ferrell, R.E., and Brufsky, A.M. 1999. Comprehensive transcript analysis in small quantities of mRNA by SAGE‐lite. Nucleic Acid Res. 27:E39.
  Velculescu, V.E., Zhang, L., Vogelstein, B., and Kenzler, K.W. 1995. Serial analysis of gene expression. Science 270:484‐487.
  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.
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