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Abstract
Objective: To optimize the use of the fluorochromic PicoGreen assay for the sensitive detection and quantification of double stranded (ds) DNA in plasma/serum samples of patients with systemic lupus erythematosus (SLE).
Methods: Plasma/serum samples were obtained from SLE patients and normal healthy donors (NHD). Plasma/serum proteins were digested with proteinase K. DNA was subsequently purified using silica-based ion exchange micro columns and detected using the PicoGreen assay.
Results: Sensitive detection of plasma/serum DNA is impaired by proteins: (1) Proteins caused background fluorescence in the PicoGreen assay. (2) Packaging of dsDNA in nucleosomes markedly reduced PicoGreen fluorescence. Therefore, we digested proteins using proteinase K and purified DNA before detection by PicoGreen assay. This procedure resulted in a detection limit for plasma/serum dsDNA of less than 1 ng/ml, and is therefore markedly more sensitive than previously described methods. We found that DNA concentrations are higher in serum than in plasma of healthy donors, suggesting artifactual DNA release during coagulation. In addition, we found higher levels of DNA in plasma and serum of a group of SLE patients compared to NHD.
Conclusions: We have optimized the use of the PicoGreen assay for the ultrasensitive and reliable quantification of DNA in plasma/serum samples. This new method can be used in future studies to explore a possible correlation between circulating DNA levels and disease activity in patients with SLE.
Acknowledgements
This work was supported by the Interdisciplinary Center for Clinical Research (IZKF, project number N2). J. A. Chen received a fellowship from the Fulbright Association.