AFLP‐Based Transcript Profiling

Pieter Vos1, Patrick Stanssens1

1 Keygene N.V., The Netherlands
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25B.5
DOI:  10.1002/0471142727.mb25b05s57
Online Posting Date:  February, 2002
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Abstract

This unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP‐fingerprinting of double‐stranded cDNA. The protocol described includes the following steps: the isolation of poly(A)+ RNA from total RNA, the synthesis of double‐stranded cDNA, the preparation of template fragments by digestion of the cDNA library with a combination of two restriction enzymes and the ligation of adaptors to the fragment ends, the selective amplification of specific subsets of fragments, and the electrophoretic analysis of these amplification products on standard denaturing polyacrylamide gels. The transcript profiles obtained by this technique are a reliable and efficient tool to identify differentially expressed mRNAs.This unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP‐finger his unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP‐finger

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Total RNA (unit 4.2 or equivalent)
  • 5′‐biotinylated oligo‐dT 25 (5‐biotin‐dT 25)
  • recipe1× and 2× binding buffer (see recipe)
  • H 2O: Milli‐Q purified (i.e., water deionized by passage through a five‐stage Milli‐Q Plus system; Millipore) or double‐distilled
  • Streptavidin‐coated magnetic beads (Dynal)
  • recipeWash buffer (see recipe)
  • 2 mM EDTA, pH 7.5
  • recipe5× first‐strand buffer (see recipe)
  • recipe5× second‐strand buffer (see recipe)
  • 0.1 M DTT ( appendix 22)
  • 5 and 10 mM (each) mixture of all 4 dNTPs (Pharmacia or unit 3.4)
  • SuperScript II (Life Technologies)
  • E. coli DNA ligase (Life Technologies)
  • E. coli DNA polymerase I (Pharmacia Biotech)
  • RNase H (Pharmacia Biotech)
  • recipe2× and 1× STEX (see recipe)
  • 10 mM Tris⋅Cl, pH 8.0/ 0.1 mM EDTA ( appendix 22)
  • TaqI restriction endonuclease (New England Biolabs; unit 3.1)
  • recipe5× RL buffer (see recipe)
  • MseI restriction endonuclease (New England Biolabs; unit 3.1)
  • 50 pmol/µl TaqI adapter top and bottom strands (see recipe for recipeoligonucleotides and double‐stranded adapters)
  • 50 pmol/µl MseI adapter top and bottom strands (see recipe for recipeoligonucleotides and double‐stranded adapters)
  • 10 mM ATP (Pharmacia)
  • T4 DNA ligase (Pharmacia)
  • 8 pmol/µl AFLP + 0 (nonselective) primers (see recipe for recipeoligonucleotides and double‐stranded adapters): TaqI + 0 and MseI + 0 primers
  • recipe10× PCR buffer (see recipe)
  • AmpliTaq DNA polymerase (Perkin‐Elmer; unit 3.5)
  • 10 µCi/µl (∼2000 Ci/mmol) [33P‐γ]ATP (Amersham)
  • recipe10× T4 polynucleotide kinase buffer (see recipe)
  • T4 polynucleotide kinase (Pharmacia; unit 3.4)
  • 8 pmol/µl AFLP +1 and + 2 (selective) primers (see recipe for recipeoligonucleotides and double‐stranded adapters): TaqI + 1 and + 2 and MseI + 1 and + 2 primers
  • AmpliTaq‐Gold polymerase (Perkin‐Elmer)
  • recipeLoading dye (see recipe)
  • Repel silane (Pharmacia)
  • Bind silane solution, fresh: Combine 30 µl bind silane (Pharmacia Biotech) and 30 µl glacial acetic acid in 10 ml ethanol
  • recipe4.5% denaturing polyacrylamide gels (see recipe)
  • recipe1× TBE (see recipe)
  • Molecular weight standard (e.g., SequaMark 10‐base ladder; Research Genetics; optional)
  • 10% acetic acid
  • Microcentrifuge tubes, RNase free
  • Magnetic plate chamber (MPC; Dynal)
  • PE‐9600 thermal cycler (Perkin Elmer) and PCR microtiter plate
  • Sequencing gel system (e.g., BioRad 38 × 50 × 0.04–cm SequiGen sequencing gel system)
  • PhosphorImager (Fujix BAS 2000, Molecular Dynamics STORM 824)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5), analysis by denaturing polyacrylamide gel electrophoresis (unit 2.12), and detection of DNA by autoradiography or phosphor imaging ( appendix 3A)
NOTE: Suppliers and brands are generally not very critical, however in case of problems it is advised to use the suggested suppliers for at least the reverse transcriptase (SuperScript II) and Taq polymerases (AmpliTaq and AmpliTaq‐Gold).NOTE: When preparing AFLP amplifications, it is advisable to work with mixes of reagents as much as possible. Working with mixes facilitates assembly and is also important for the reliability and reproducibility of the reactions. In practice, the assembly of the mixes depends on the experiment—i.e., which components remain constant in a series of reactions: the template‐DNA or the primer combinations (e.g., one sample with many primer combinations, many samples with one primer combination).
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Figures

Videos

Literature Cited

Literature Cited
   Adams, M.D., Kelley, J.M., Gocayne, J.D., Dubnick, M., Polymeropoulos, M.H., Xiao, H., Merril, C.R., Wu, A., Olde, B., Moreno, R.F., Kervalage, A.R., McCombie, W.R., and Venter, J.G. 1991. Complementary DNA sequencing: Expressed sequence tags and the human genome project. Science 252:1651‐1655.
   Bachem, C.W.B., Van der Hoeven, R.S., De Bruijn, S.M., Vreugdenhil, D., Zabeau, M., and Visser, R.G.F. 1996. Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber development. Plant J. 9:745‐753.
   Brenner, S., Johnson, M., Bridgham, J., Golda, G., Lloyd, D.H., Johnson, D., Luo, S., McCurdy, S., Foy, M., Ewan, M., Roth, R., George, D., Eletr, S., Albrecht, G., Vermanas, E., Williams, S.R.R., Moon, K., Burcham, T., Pallas, M., DuBridge, R.B., Kirchner, J., Fearson, K., Mao, J., and Corcoran, K. 2000. Gene expression analysis by massively parallel signature sequencing on microbead arrays. Nat. Biotechn. 18:630‐634.
   Breyne, P. and Zabeau, M. 2001. Genome‐wide expression analysis of plant cell cycle modulated genes. Curr. Opin. Plant Biol. 4:136‐142.
   De Risi, J.L., Iyer, V.R., and Brown, P.O. 1997. Exploring the metabolic and genetic control of gene expression on a genome scale. Science 278:1359‐1367.
   Din, R.F., Nesert, E.W., and Comai, L. 2001. Plant gene expression response to Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. U.S.A. 98:10954‐10959.
   Durrant, W.E., Rowland, O., Piedras, P., Hammond‐Kosack, K.E., and Jones, J.D. 2000. cDNA‐AFLP reveals a striking overlap in race‐specific resistance and wound response gene expression profiles. Plant Cell 12:963‐977.
   Dynal. 1995. Biomagnetic techniques in molecular biology. Technical Handbook, Second Edition. Dynal A.S, Oslo, Norway.
   Fischer, A., Saedler, H., and Theissen, G. 1995. Restriction fragment length polymorphism‐coupled domain‐directed differential display: A highly efficient technique for expression analysis of multigene families. Proc. Natl. Acad. Sci. U.S.A. 92:5331‐5335.
   Liang, P. and Pardee, A.B. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967‐971.
   Prashar, Y. and Weismann, S.M. 1996. Analysis of differential gene expression by display of 3′ end restriction fragments of cDNAs. Proc. Natl. Acad. Sci. 93:659‐663.
   Qin, L., Prins, P., Jones, J.T., Popeijus, J., Smant, G., Bakker, J., and Helder, J. 2001. GenEst, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA‐AFLP. Nucl. Acids. Res. 29:1616‐1622.
   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.
   Van der Biezen, E.A., Juwana, H., Parker, J.E., and Jones, J.D. 2000. cDNA‐AFLP reveals a striking overlap in race‐specific resistance and wound response gene expression profiles. Plant Cell. 12:963‐977.
   Velculescu, V., Zhang, L., Vogelstein, B., and Kinzler, K.W. 1995. Serial analysis of gene expression. Science 270:484‐487.
   Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., and Zabeau, M. 1995. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res. 23:4407‐4414.
   Vos, P. and Kuiper, M. 1998. AFLP analysis. In DNA Markers: Protocols, Applications and Overviews (G. Caetano‐Anolles and P.M. Gresshoff, eds.) pp. 115‐131. John Wiley and Sons, New York.
   Welsh, J.B., Zarrinkar, P.P., Supinosos, L.M., Kern, S.G., Behling, C.A., Monk, B.J., Lockhart, D.J., Burger, S.A., and Hampton, G.M. 2001. Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer. Proc. Natl. Acad. Sci. 98:1176‐1181.
   Wodicka, L., Dong, H., Millmann, M., Ho, M.H., and Lockhart, D.J. 1997. Genome‐wide expression monitoring in Saccharomyces cerevisiae. Nature Genet. 15:1359‐1367.
   Zabeau, M. and Vos, P. 1993. Selective restriction fragment amplification: A general method for DNA fingerprinting. European Patent EP 0534858‐ B1.
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