Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen and Hoechst 33258.
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 (0.9ng DNA/ml) than Hoechst 33258 (6ng DNA/ml) and can also be used in a microplate reader.


CONCLUSIONS
PicoGreen can be used in a rapid and sensitive assay to quantify DNA in small samples of human cartilage.
Department of Bioengineering and Institute for Biomedical Engineering, University of California, San Diego, La Jolla 92093-0412, USA.
K B
KB
McGowan
M S
MS
Kurtis
L M
LM
Lottman
D
D
Watson
R L
RL
Sah
D000328:Adult; D000368:Aged; D001562:Benzimidazoles; D002358:Cartilage, Articular / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D019286:Endopeptidase K; D005456:Fluorescent Dyes; D005470:Fluorometry; D006801:Humans; D008875:Middle Aged; D009300:Nasal Septum / Q000737:chemistry*; D009930:Organic Chemicals; D013048:Specimen Handling / Q000379:methods
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; C447680:hoechst 32258; D004247:DNA; D019286:Endopeptidase K