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Related Experiment Videos

A method for the absolute quantification of cDNA using real-time PCR.

Joseph A Whelan1, Nick B Russell, Michael A Whelan

  • 1Onyvax Ltd., St. George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK.

Journal of Immunological Methods
|September 6, 2003
PubMed
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This study introduces a novel real-time PCR method for precise absolute quantification of cDNA. The technique uses a plasmid reference curve and DNA quantification, enabling standardized, comparable results across experiments for gene expression analysis.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Real-time PCR (polymerase chain reaction) is a powerful gene expression analysis tool.
  • Current data presentation lacks standardization, leading to interpretation challenges.
  • Reliance on non-standard calibration genes introduces variability in experimental results.

Purpose of the Study:

  • To develop a novel, standardized method for absolute quantification of cDNA species using real-time PCR.
  • To overcome limitations in current data presentation conventions for real-time PCR.
  • To enable reliable inter- and intra-experimental comparisons of gene expression levels.

Main Methods:

  • Utilized accurate double-stranded DNA quantification with PicoGreen and fluorescence.

Related Experiment Videos

  • Developed a plasmid reference curve using cloned real-time PCR products.
  • Employed the same primers for PCR amplification and subsequent plasmid cloning.
  • Expressed results as copy number per microgram of oligo-dT primed cDNA.

    • Main Results:

    • Successfully quantified absolute levels of interferon-gamma, TNF-alpha, interleukin-2, and interleukin-10 in a human system.
    • Demonstrated remarkable correlation between measured gene copy numbers and released protein levels.
    • Established a reproducible method for absolute gene quantification.

    Conclusions:

    • The novel method provides a standardized approach for absolute quantification in real-time PCR.
    • This technique enhances the reliability and comparability of gene expression data.
    • The method is broadly applicable to various real-time PCR applications beyond cytokine quantification.