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Related Experiment Videos

Total cellular RNA content: correlation between flow cytometry and ultraviolet spectroscopy.

K D Bauer, L A Dethlefsen

    The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
    |June 1, 1980
    PubMed
    Summary

    Acridine orange staining accurately quantifies total cellular RNA using flow cytometry. This method shows a strong correlation with ultraviolet spectroscopy, despite a minor DNA fluorescence contribution.

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    Area of Science:

    • Cell Biology
    • Biochemistry
    • Spectroscopy

    Background:

    • Accurate quantification of cellular RNA is crucial for molecular biology research.
    • Ultraviolet (UV) spectroscopy is a standard method for determining RNA concentration.
    • Flow cytometry offers high-throughput analysis of cellular components.

    Purpose of the Study:

    • To compare the accuracy of acridine orange staining and flow cytometry for quantifying total RNA with UV spectroscopy.
    • To evaluate the reliability of acridine orange as a fluorescent probe for RNA in cellular analysis.

    Main Methods:

    • Chinese hamster ovary and HeLa-S3 cells were used for the study.
    • Total RNA content was determined using ultraviolet spectroscopy.
    • Cellular RNA was stained with acridine orange and analyzed by flow cytometry.

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  • RNAse treatments were applied to assess specificity.

    • Main Results:

    • A high correlation coefficient (0.93) was observed between RNA content measured by UV spectroscopy and acridine orange staining analyzed via flow cytometry.
    • Acridine orange staining demonstrated effectiveness in quantifying total cellular RNA.
    • A low but significant red fluorescence background from DNA was detected.

    Conclusions:

    • Acridine orange staining coupled with flow cytometry provides a reliable and effective method for quantitating total cellular RNA.
    • This technique is a viable alternative to UV spectroscopy for RNA content estimation in cell populations.
    • Researchers should be aware of the potential DNA-derived background fluorescence.