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

Abstract

Objective: To compare two fluorometric assays, utilizing (1) the bisbenzimidazole Hoechst 33258 and (2) PicoGreene®, 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. © 2002 Published by Elsevier Science Ltd on behalf of OsteoArthritis Research Society International.