Restriction‐Mediated Differential Display (RMDD)

Achim Fischer1

1 F. Hoffmann‐La Roche AG, Basel, Switzerland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25B.4
DOI:  10.1002/0471142727.mb25b04s56
Online Posting Date:  November, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

A variation on differential display, restriction‐mediated differential display (RMDD) presents an alternative approach to the fragment display technologies. It touts high sensitivity (detection of mRNAs at a dilution of 1:100,000 and of regulation factors lower than two‐fold), a strategy for avoiding false positives, nonradioactive detection, and universal applicability to any polyadenylated RNA.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: RMDD Library Preparation and Two‐Round Amplification
  • Alternate Protocol 1: Amplification by Two‐Phase PCR
  • Support Protocol 1: Direct Blotting Electrophoresis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: RMDD Library Preparation and Two‐Round Amplification

  Materials
  • 50 µg total RNA (units 4.1 & 4.2)
  • RNase‐free water
  • 10 µM cDNA primer CP29V: 5′‐ACC TAC GTG CAG ATT TTT TTT TTT TTT TX 1‐3′ (X 1 = A, C, or G; equimolar amounts of all three species; see unit 2.11 for oligonucleotide synthesis)
  • 100 mM RNase‐free DTT (Life Technologies)
  • 5× SuperScript buffer (Life Technologies)
  • 10 mM RNase‐free and standard dNTPs
  • 40 U/µl RNase inhibitor (e.g., RNasin)
  • 200 U/µl SuperScript II reverse transcriptase (Life Technologies)
  • 5× second‐strand buffer II (unit 5.5)
  • 1.5 U/µl RNase H
  • 10 U/µl E. coli DNA polymerase I
  • Phenol equilibrated with TE buffer, pH 8.0 (unit 2.1)
  • Chloroform
  • 20 mg/ml glycogen
  • recipe28% PEG 8000/3.6 mM MgCl 2 (see recipe)
  • 70% and 100% ethanol
  • 10× universal buffer (Stratagene)
  • 4 U/µl MboI restriction endonuclease (Stratagene)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 10 mM ATP
  • recipe0.5 µg/µl MboI‐linker ML2025 (see recipe)
  • T4 DNA ligase and 10× buffer (Roche)
  • 1× and 0.25× TE buffer, pH 8.0 ( appendix 22)
  • 4 µM primer CP28X 1: 5′‐ACC TAC GTG CAG ATT TTT TTT TTT TTT TX 1‐3′ (X 1 = A, C, or G; see unit 2.11 for oligonucleotide synthesis)
  • 4 µM primer ML19Y 1: 5′‐TGC TAA GTC TCG CGA GAT CY 1‐3′ (Y 1 = A, C, G, or T; see unit 2.11 for oligonucleotide synthesis)
  • recipe10× PCR buffer (see recipe)
  • 20 mM MgCl 2 ( appendix 22)
  • RediLoad (Research Genetics)
  • 5 U/µl Taq DNA polymerase
  • 100‐bp DNA size ladder (e.g., Life Technologies)
  • 1.5% agarose gel (unit 2.5)
  • 4 µM primer CP28X 1X 2: 5′‐ACC TAC GTG CAG ATT TTT TTT TTT TTT T X 1X 2‐3′ (X 2 = A, C, G, or T; see unit 2.11 for oligonucleotide synthesis)
  • 4 µM labeled primer *ML18Y 1Y 2: 5′‐*GCT AAG TCT CGC GAG ATC Y 1Y 2‐3′ (Y2 = A, C, G, or T; see unit 2.11 for oligonucleotide synthesis)
  • Formamide buffer: 5 mM EDTA/0.1% bromophenol blue in 99% deionized formamide
  • 22°, 37°, 42°, 65° and 75°C water bath, heat blocks, or equivalent
  • Thermal cycler with heated lid
  • 96‐well PCR plates (e.g., MJ Research)
  • Additional reagents and equipment for ethanol precipitation and phenol/chloroform extraction of DNA (unit 2.1), and pouring and running (unit 2.5) agarose and 6% polyacrylamide gels (unit 7.6)

Alternate Protocol 1: Amplification by Two‐Phase PCR

  • 0.1 mM dNTPs (freshly diluted from 10 mM dNTPs)

Support Protocol 1: Direct Blotting Electrophoresis

  • TBE electrophoresis buffer ( appendix 22) standard and degassed (i.e., stirred under vacuum 20 min)
  • recipeMaleic buffer, pH 7.5 (see recipe)
  • recipe1.5% blocking reagent (see recipe)
  • Streptavidin‐alkaline phosphatase conjugate (Roche Molecular Biochemicals)
  • recipeReaction buffer, pH 9.5 (see recipe)
  • NBT/BCIP in 67% (v/v) DMSO (Roche Molecular Biochemicals)
  • Primers (see unit 2.11 for oligonucleotide synthesis):
  •  CP28: 5′‐ACC TAC GTG CAG ATT TTT TTT TTT TTT T‐3′
  •  ML18: 5′‐GCT AAG TCT CGC GAG ATC‐3′
  • GATC 1500 Direct Blotting Electrophoresis System (GATC Biotech AG)
  • Direct blotting membrane (GATC Biotech AG)
  • 10‐ml syringe and 25‐G needle
  • 32‐well sharkstooth comb
  • GELoader tips (Eppendorf) with capillary‐like part cut away
  • Stratalinker (Stratagene)
  • Developing drum (e.g., GATC tube; GATC Biotech AG)
  • Adhesive tape
  • Rolling incubator accepting 18 × 35–cm tubes and capable of revolving at ∼20 rpm
  • 2‐mm‐thick polyethylene wrap (e.g., Neolab, Heidelburg, FRG) or material from a thick hybridization bag
  • T‐A cloning system (e.g., Invitrogen; optional)
  • Additional reagents and materials for casting denaturing polyacrylamide gels (unit 2.12), agarose gel electrophoresis (unit 2.5), and molecular cloning of PCR products (unit 15.7).
NOTE: For details concerning use of the GATC 1500 Direct Blotting Electrophoresis apparatus, consult the manufacturer's instructions.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Beck, S. and Pohl, F.M. 1984. DNA sequencing with direct blotting electrophoresis. EMBO J. 3:2905‐2909.
   Debouck, C. 1995. Differential display or differential dismay? Curr. Opin. Biotechnol. 6:597‐599.
   Fischer, A. 1995. Verfahren zur Genexpressionsanalyse. German patent application DE 195 18 505.6 [other members of the same patent family are given in the introduction].
   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.
   Karrer, E.E., Lincoln, J.E., Hogenhout, S., Bennett, A.B., Bostock, R.M., Martineau, B., Lucas, W.J., Gilchrist, D.G., and Alexander, D. 1995. In situ isolation of mRNA from individual plant cells: Creation of cell‐specific cDNA libraries. Proc. Natl. Acad. Sci. U.S.A. 92:3814‐3818.
   Ledakis, P., Tanimura, H., and Fojo, T. 1998. Limitations of differential display. Biochem. Biophys. Res. Commun. 251:653‐656.
   Liang, P. and Pardee, A.B. 1992. Differential display of eucaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967‐971.
   Malhotra, K., Foltz, L., Mahoney, W.C., and Schueler, P.A. 1998. Interaction and effect of annealing temperature on primers used in differential display RT‐PCR. Nucl. Acids Res. 26:854‐856.
   McClelland, M. and Welsh, J. 1994. RNA fingerprinting by arbitrarily primed PCR. PCR Methods Appl. 4:S66‐S81.
   Poirier, G.M., Pyati, J., Wan, J.S., and Erlander, M.G. 1997. Screening differentially expressed cDNA clones obtained by differential display using amplified RNA. Nucl. Acids Res. 25:913‐914.
   Prashar, Y. and Weissman, S.M. 1996. Analysis of differential gene expression by display of 3′ end restriction fragments of cDNAs. Proc. Natl. Acad. Sci. U.S.A. 93:659‐663.
   Shimkets, R.A., Lowe, D.G., Tai, J.T., Sehl, P., Jin, H., Yang, R., Predki, P.F., Rothberg, B.E., Murtha, M.T., Roth, M.E., Shenoy, S.G., Windemuth, A., Simpson, J.W., Simons, J.F., Daley, M.P., Gold, S.A., McKenna, M.P., Hillan, K., Went, G.T., and Rothberg, J.M. 1999. Gene expression analysis by transcript profiling coupled to a gene database query. Nature Biotechnol. 17:798‐803.
   Sutcliffe, J.G., Foye, P.E., Erlander, M.G., Hilbush, B.S., Bodzin, L.J., Durham, J.T., and Hassle, K.W. 2000. TOGA: An automated parsing technology for analyzing expression of nearly all genes. Proc. Natl. Acad. Sci. U.S.A. 97:1976‐1981.
   Trentmann, S.M. 1995. Alternatives to 35S as a label for the differential display of eucaryotic messenger RNA. Science 267:1186.
   Welsh, J., Chada, K., Dalal, S.S., Cheng, R., Ralph, D., and McClelland, M. 1992. Arbitrarily primed PCR fingerprinting of RNA. Nucl. Acids Res. 20:4965‐4970.
   Zhao, S., Ooi, S.L., and Pardee, A.B. 1995. New primer strategy improves precision of differential display. Biotechniques 18:842‐846, 848, 850.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library