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Related Experiment Videos

RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization

L J Jones1, S T Yue, C Y Cheung

  • 1Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, Oregon, 97402-9165, USA.

Analytical Biochemistry
|January 12, 1999
PubMed
Summary
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A new RiboGreen RNA quantitation reagent offers a highly sensitive fluorescence assay for RNA detection. This method surpasses existing techniques, enabling precise RNA quantification across various biological samples.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Accurate RNA quantification is crucial for molecular biology research.
  • Existing RNA detection methods often lack sensitivity or are complex.
  • Development of a sensitive and user-friendly RNA assay is needed.

Purpose of the Study:

  • To develop and characterize a novel fluorescence-based assay for sensitive RNA quantitation.
  • To evaluate the performance of the RiboGreen RNA quantitation reagent.
  • To establish the assay's applicability in diverse experimental conditions.

Main Methods:

  • Development of a fluorescence-based solution assay utilizing the RiboGreen RNA quantitation reagent.
  • Characterization of the dye's fluorescence properties upon nucleic acid binding.

Related Experiment Videos

  • Assessment of assay sensitivity, linear quantitation range, and robustness in the presence of common biochemicals.

    • Main Results:

    • RiboGreen reagent demonstrates >1000-fold fluorescence enhancement upon RNA binding.
    • The assay achieves a detection limit of 1.0 ng/ml RNA, exceeding ethidium bromide sensitivity by 200-fold.
    • Linear RNA quantitation over three orders of magnitude (1.0 ng/ml to 1.0 µg/ml) was achieved with high correlation (r² = 0.999).
    • Assay linearity and sensitivity were maintained in the presence of various salts, proteins, and other common laboratory reagents.

    Conclusions:

    • The RiboGreen assay provides a highly sensitive, rapid, and robust method for RNA quantitation.
    • This assay is adaptable for various RNA types and compatible with standard fluorescence instrumentation.
    • The RiboGreen assay is suitable for high-throughput applications and automation in molecular biology.