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Related Experiment Videos

Transcript quantitation in total yeast cellular RNA using kinetic PCR.

J J Kang1, R M Watson, M E Fisher

  • 1Department of Biological Chemistry, School of Medicine, University of California, 1 Shields Avenue, Davis, CA 95616, USA.

Nucleic Acids Research
|December 22, 1999
PubMed
Summary
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Kinetically monitored, reverse transcriptase-initiated PCR (kRT-PCR) offers a sensitive and accurate method for high-throughput transcript quantitation. This novel assay provides advantages over existing technologies for analyzing RNA levels in various cell states.

Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • High-throughput transcript quantitation is crucial for understanding cellular processes.
  • Existing methods like DNA microarrays and SAGE have limitations in certain applications.
  • Novel assays are needed for efficient and accurate RNA analysis.

Purpose of the Study:

  • To introduce and evaluate kinetically monitored, reverse transcriptase-initiated PCR (kRT-PCR) for high-throughput transcript quantitation.
  • To assess the reproducibility, sensitivity, and accuracy of kRT-PCR.
  • To compare kRT-PCR with existing technologies for RNA analysis.

Main Methods:

  • Kinetically monitored, reverse transcriptase-initiated PCR (kRT-PCR) was applied to total cellular RNA.
  • Yeast transcripts, previously quantified by SAGE analysis, were used for validation.

Related Experiment Videos

  • Assay performance was assessed across a wide range of transcript abundance.

    • Main Results:

    • kRT-PCR demonstrated reproducible quantification of transcript level changes between different cell states (+/-20% accuracy).
    • The assay exhibited high sensitivity, quantifying transcripts over five orders of magnitude.
    • Low abundance transcripts, including those for cell cycle and transcriptional regulators, were successfully quantified.

    Conclusions:

    • kRT-PCR is a novel, sensitive, and accurate method for high-throughput transcript quantitation.
    • The assay offers advantages in simplicity and flexibility compared to DNA microarrays and SAGE.
    • kRT-PCR is suitable for analyzing transcript levels in diverse genetic and physiological conditions.