Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen® and Hoechst 33258

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McGowan, K. B.¹; Kurtis, M. S.¹; Lottman, L. M.¹; Watson, D.²; Sah, R. L.¹

Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen^® and Hoechst 33258

Osteoarthritis Cartilage. July 2002;10(7):580-587

©2002 Published by Elsevier Science Ltd on behalf of OsteoArthritis Research Society International.

Affiliations

¹Department of Bioengineering and Institute for Biomedical Engineering, University of California, San Diego, La Jolla, CA, U.S.A.

²Division of Head and Neck Surgery, University of California, San Diego, La Jolla, CA, U.S.A.
Abstract and keywords

Objective: To compare two fluorometric assays, utilizing (1) the bisbenzimidazole Hoechst 33258 and (2) PicoGreen^®, for determining DNA content in human cartilage.

Methods: Human articular and nasal septal cartilage explants were digested using proteinase K. Portions of sample digest were analysed for intrinsic and dye-enhanced fluorescence with either Hoechst 33258 or PicoGreen^®.

Results: Intrinsic tissue fluorescence in both articular and septal cartilage increased with age and was prominent at wavelengths used for Hoechst 33258 but relatively low at wavelengths used for PicoGreen^®. The relative contribution of intrinsic fluorescence to total dye-enhanced fluorescence of human cartilage was markedly greater for Hoechst 33258 (19–57%) than for PicoGreen^® (2–7%). Thus, in many situations, DNA in human cartilage can be assayed using PicoGreen^® without the need to correct for intrinsic cartilage fluorescence. The enhancement of fluorescence by each dye was found to be specific for DNA, as shown by fluorescence spectra, >90% sensitivity to DNase, and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in the absence or presence of proteinase K digested human cartilage, once intrinsic cartilage fluorescence was subtracted. PicoGreen^® was more sensitive for assaying DNA (0.9 ng DNA/ml) than Hoechst 33258 (6 ng DNA/ml) and can also be used in a microplate reader.

Conclusion: PicoGreen^® can be used in a rapid and sensitive assay to quantify DNA in small samples of human cartilage.

Keywords: Human cartilage, DNA, Fluorescence, Hoechst 33258, PicoGreen®
Links

DOI: 10.1053/joca.2002.0794

PubMed: 12127839

WoS: 000177625700011
History

Received: 2001-06-7

Accepted: 2002-03-18

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