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Related Experiment Videos

Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR.

M S Rajeevan1, S D Vernon, N Taysavang

  • 1Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, Atlanta, GA 30333, USA. mor4@cdc.gov

The Journal of Molecular Diagnostics : JMD
|February 28, 2001
PubMed
Summary

Real-time reverse transcription-polymerase chain reaction (RT-PCR) effectively validates gene expression changes identified by DNA arrays. This method confirms differential gene expression, crucial for accurate biological research.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • DNA arrays are used to identify differentially expressed genes.
  • Keratinocyte subclones with different human papillomavirus states serve as a model system.
  • Validating array findings is essential for reliable gene expression analysis.

Purpose of the Study:

  • To evaluate real-time (kinetic) reverse transcription-polymerase chain reaction (RT-PCR) for validating DNA array results.
  • To assess the reliability of DNA array data based on hybridization intensity and differential expression levels.
  • To determine the suitability of LightCycler-based real-time RT-PCR for gene expression validation.

Main Methods:

  • Utilized high-density filter arrays to screen gene expression between two keratinocyte subclones.

Related Experiment Videos

  • Employed real-time RT-PCR with LightCycler-based SYBR Green I dye detection and melting curve analysis.
  • Quantified relative gene expression changes for validation.

    • Main Results:

    • High-density filter arrays identified 144 genes with twofold or greater expression difference out of 588 genes.
    • Real-time RT-PCR confirmed the expression change in 71% (17 of 24) of the genes identified by arrays.
    • Genes with strong hybridization signals and at least twofold difference were more likely to be validated.

    Conclusions:

    • Hybridization intensity and differential expression level influence DNA array result validation.
    • Real-time RT-PCR is a quantitative, rapid, and RNA-efficient method for validating DNA array findings.
    • LightCycler technology is well-suited for validating DNA array results, confirming gene expression changes accurately.