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Related Experiment Videos

Increased precision of microbial RNA quantification using NASBA with an internal control.

Stacey S Patterson1, Erica T Casper, Luis Garcia-Rubio

  • 1College of Marine Science, University of South Florida, 140 7th Ave., South, St. Petersburg, FL 33701, USA.

Journal of Microbiological Methods
|January 15, 2005
PubMed
Summary

This study introduces a novel method using a synthetic internal control RNA (IC-RNA) to improve the accuracy and precision of nucleic acid based sequence amplification (NASBA) for low abundance RNA detection. The new approach enhances quantification reliability in various applications.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Environmental Science

Background:

  • Detection and quantification of low abundance RNA are crucial for diagnostics, environmental monitoring, gene expression, and biodefense.
  • Nucleic acid based sequence amplification (NASBA) offers high sensitivity but can suffer from variability due to its multi-enzyme requirement.
  • Existing NASBA methods may exhibit greater variability compared to PCR-based assays.

Purpose of the Study:

  • To enhance the accuracy and precision of RNA quantification using NASBA.
  • To overcome the inherent variability in traditional NASBA assays.
  • To develop a robust method for quantifying any RNA sequence, including low abundance targets.

Main Methods:

  • Adaptation of the bioMérieux Nuclisens Basic Kit and EasyQ Analyzer.

Related Experiment Videos

  • Introduction of a synthetic internal control RNA (IC-RNA) for improved quantification.
  • Quantification of the rbcL gene from Karenia brevis using a ratio of time to positivity (TTP) for wild-type and IC-RNA.

    • Main Results:

    • A simple method was developed to accurately quantify native target RNA.
    • The ratio of TTP values for wild-type and IC-RNA against starting target molecules improved quantification.
    • Significantly increased accuracy and precision in prediction compared to standard TTP calculations were achieved.

    Conclusions:

    • The developed method using IC-RNA significantly enhances the accuracy and precision of NASBA-based RNA quantification.
    • This approach offers a reliable solution for detecting and quantifying low abundance RNA targets in diverse applications.
    • The method provides a more robust alternative to standard TTP calculations for sensitive RNA detection.