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DNA Assay

Deoxyribonucleic acid (DNA) is a distinct cell component, therefore DNA amounts are correlated with cell numbers (1).  For example, in cartilage tissue engineering, artificial cartilage quality is evaluated by DNA amounts translated as chondrocyte numbers, as well as amounts of collagen and glycosaminoglycans (GAGs) in extracellular matrix components (ECM)(2, 3).  DNA can be quantified by the ratio of absorbance at 260 nm and 280 nm, however, this method is affected by contaminating proteins, RNA and chemicals in the sample, thus requiring additional DNA isolation steps.  For tissue analysis, Chondrex provides an alternative DNA assay kit employing the Hoechst 33258 fluorescent dye which specifically binds to Adenine-Thymine base pairs, resulting in fluorescence at excitation 360 nm/emission 460 nm.   Since the dye-DNA binding and the fluorescence intensity are unaffected by contaminating proteins and other substances  in an optimized assay condition, this DNA assay kit works accurately with samples containing other analytes.

Chondrex also provides native collagen detection ELISAs (Catalog # 6012-6015, 6018 and 6021), Hydroxyproline assay kit (Catalog # 6017), Sirius red/ fast green staining kit (Catalog # 9046 ), Sirius red total collagen detection kit (Catalog # 9062) and GAGs assay kit (Catalog # 6022).  These kits will facilitate further analysis of your studies. 

DNA Assay Kit

Product Quantity Catalog # Price (USD)
DNA Assay Kit 1 Kit 6023 90.00

Table 1 provides a useful matrix to determine the ideal solubilization protocol for the analyte of interest.  In most cases, two different protocols will need to be used.  For example, protocol A extracts GAGs, but fails to completely extract collagen and DNA, resulting in underestimated values (4,5).  Protocol B solubilizes GAGs (6) and DNA (7), but degrades collagen.  Finally, protocol C (Chondrex’s protocol) can be used to measure GAGs and collagen (8), but not DNA.

REFERENCES

1.  V. M. Quent, D. Loessner, T. Friis, J. C. Reichert, D. W. Hutmacher, Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research. J Cell Mol Med 14, 1003-1013 (2010).

2.  J. H. Yoon, J. Halper, Tendon proteoglycans: biochemistry and function. J Musculoskelet Neuronal Interact 5, 22-34 (2005).

3.  J. A. Buckwalter, H. J. Mankin, Articular cartilage: tissue design and chondrocyte-matrix interactions. Instr Course Lect 47, 477-486 (1998).

4.  C. D. Hoemann, J. Sun, V. Chrzanowski, M. D. Buschmann, A multivalent assay to detect glycosaminoglycan, protein, collagen, RNA, and DNA content in milligram samples of cartilage or hydrogel-based repair cartilage. Anal Biochem 300, 1-10 (2002).

5.  S. W. Sajdera, V. C. Hascall, Proteinpolysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures. J Biol Chem 244, 77-87 (1969).

6.  R. W. Farndale, D. J. Buttle, A. J. Barrett, Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochem Biophys Acta 883, 173-177 (1986).

7.  Y. J. Kim, R. L. Sah, J. Y. Doong, A. J. Grodzinsky, Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 174, 168-176 (1988).

8.  K. von der Mark, M. van Menxel, H. Wiedemann, Isolation and characterization of new collagens from chick cartilage. Eur J Biochem 124, 57-62 (1982).