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Cloning Differentially Expressed Genes Using Rapid Subtraction Hybridization (RaSH)

  • Habib Boukerche
  • Zao-zhong Su
  • Dong-chul Kang
  • Paul B. Fisher
Part of the Methods in Molecularbiology™ book series (MIMB, volume 383)

Abstract

Differential gene expression represents the entry point for comprehending complex biological processes. In this context, identification and cloning of differentially expressed genes represent critical elements in this process. Many techniques have been developed to facilitate achieving these objectives. Although effective in many situations, most currently described approaches are not trouble-free and have limitations, including complexity of performance, redundancy of gene identification (reflecting cloning biases) and false-positive gene identification. A detailed methodology to perform a rapid and efficient cloning approach, called rapid subtraction hybridization is described in this chapter. This strategy has been applied successfully to a number of cell culture systems and biological processes, including terminal differentiation and cancer progression in human melanoma cells, resistance or sensitivity to HIV-1 in human T cells and gene expression changes following infection of normal human fetal astrocytes with HIV-1 or treatment with neutrotoxic agents. Based on its simplicity of performance and high frequency of genuine differential gene identification, the rapid subtraction hybridization (RaSH) approach will allow wide applications in diverse systems and biological contexts.

Key Words

RaSH gene cloning terminal differentiation metastasis reverse Northern Northern blotting 

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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Habib Boukerche
    • 1
    • 2
  • Zao-zhong Su
    • 3
  • Dong-chul Kang
    • 4
  • Paul B. Fisher
    • 1
    • 3
    • 5
    • 6
  1. 1.Department of PathologyColumbia University Medical Center, College of Physicians and SurgeonsNew York
  2. 2.INSERMLyonFrance
  3. 3.Department of UrologyColumbia University Medical Center, College of Physicians and SurgeonsNew, York
  4. 4.Ilsong Institute of Life ScienceHallym UniversityAnyang, Kyeonggi-doRepublic of Korea
  5. 5.Herbert Irving Comprehensive Cancer CenterCollege of Physicians and Surgeons, Columbia University Medical CenterNew York
  6. 6.Department of NeurosurgeryCollege of Physicians and Surgeons, Columbia University Medical CenterNew York

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