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Related Experiment Videos

Representational difference analysis of cDNA.

Lucas D Bowler1

  • 1Trafford Centre for Medical Research, University of Sussex, Brighton, UK.

Methods in Molecular Medicine
|February 13, 2004
PubMed
Summary
This summary is machine-generated.

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This chapter details Polymerase Chain Reaction-coupled subtractive hybridization of complementary DNA (cDNA) representational difference analysis. This sensitive method identifies genes with altered expression between cell populations using minimal starting material.

Area of Science:

  • Molecular Biology
  • Genomics
  • Gene Expression Analysis

Background:

  • Gene expression profiling is crucial for understanding cellular differences.
  • Existing methods may require extensive prior knowledge or large sample amounts.
  • Representational difference analysis (RDA) offers a basis for comparative gene expression.

Purpose of the Study:

  • To describe the Polymerase Chain Reaction-coupled subtractive hybridization technique of complementary DNA representational difference analysis (cDNA RDA).
  • To highlight the method's sensitivity, cost-effectiveness, and applicability to low starting material.
  • To outline the key phases involved in performing cDNA RDA.

Main Methods:

  • Utilizes representational difference analysis (RDA) combined with Polymerase Chain Reaction (PCR).

Related Experiment Videos

  • Involves PCR generation of amplicons from RNA populations.
  • Features a two-step subtractive hybridization to enrich differentially expressed genes and deplete common sequences.

    • Main Results:

    • Enriches amplified fragments of differentially expressed genes.
    • Effectively depletes sequences common to both cell populations.
    • Yields difference products suitable for purification, cloning, and sequencing.

    Conclusions:

    • cDNA RDA is a sensitive and cost-effective technique for identifying genes with modified expression.
    • The method requires no prior genome sequence data, making it broadly applicable.
    • Suitable for analyzing gene expression changes even with very low amounts of starting material.