Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double

Van Luu-The1, Nathalie Paquet, Ezequiel Calvo

  • 1Molecular Endocrinology and Oncology Research Center, Laval University, Quebec, Canada. Van.Luu-The@crchul.ulaval.ca

Biotechniques
|February 25, 2005
PubMed
Summary

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Hedgehog signaling regulates Wolffian duct development through the primary cilium†.

Biology of reproduction·2022
Same author

Verification of Nuclear Magnetic Resonance Characterization of Traditional Homeopathically Manufactured Metal (Cuprum metallicum) and Plant (Gelsemium sempervirens) Medicines and Controls.

Homeopathy : the journal of the Faculty of Homeopathy·2020
Same author

Hedgehog signaling pathway regulates gene expression profile of epididymal principal cells through the primary cilium.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology·2020
Same author

Proteomic markers of low and high fertility bovine spermatozoa separated by Percoll gradient.

Molecular reproduction and development·2019
Same author

Toxicity of tailing leachates from a niobium mine toward three aquatic organisms.

Ecotoxicology and environmental safety·2019
Same author

The Passing of Dr. Fernand Labrie (1937-2019): The legacy of excellence in science.

The Journal of steroid biochemistry and molecular biology·2019

This study introduces a more robust real-time quantitative PCR (RT-qPCR) method for gene expression analysis. The second derivative method with double housekeeping gene correction offers improved accuracy and low variation for high-throughput measurements.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • Real-time quantitative PCR (RT-qPCR) is crucial for validating DNA microarray and GeneChip results.
  • Existing RT-qPCR quantification methods can be user-dependent and less suitable for high-throughput analysis.
  • There is a need for more robust and accurate RT-qPCR methods, especially for quantifying low gene expression levels.

Purpose of the Study:

  • To compare a user-independent second derivative method with the user-dependent fit point method for RT-qPCR.
  • To evaluate the benefits of a double correction strategy using housekeeping genes for enhanced accuracy.
  • To demonstrate the suitability of an improved RT-qPCR method for high-throughput gene expression quantification.

Main Methods:

  • Comparison of the second derivative method versus the fit point method for RT-qPCR analysis.

Related Experiment Videos

  • Implementation of a double correction technique: internal standard correction with a housekeeping gene and reference correction using external housekeeping gene levels.
  • Assessment of method efficiency and accuracy, particularly for low gene expression levels.

    • Main Results:

    • The real-time PCR method demonstrated high efficiency, with coefficients near the theoretical value of two.
    • The second derivative method proved more accurate than the fit point method for quantifying low gene expression levels.
    • The developed method exhibited low measurement variation, with coefficients of variation averaging less than 1% across triplicate experiments.

    Conclusions:

    • The second derivative method, combined with double housekeeping gene correction, provides a robust and accurate approach for RT-qPCR.
    • This improved RT-qPCR method is suitable for high-throughput gene expression analysis and validation.
    • The method minimizes errors from sample handling and variations in housekeeping gene expression across different tissues or treatments.