Determination of residual DNA in decellularised aortas- towards fluorescence-based quantification of DNA purified by various methods.

BACKGROUND: Decellularisation of animal tissues is a promising strategy to obtain scaffolds for tissue engineering. However, double-stranded (ds)DNA of animal origin that may reside in tissue even after harsh decellularisation causes adverse reactions in patients. Thus, precise determination of residual dsDNA is essential, but challenging since the methods used for purification may affect quantification. METHODS: To elucidate the best method for purifying and quantifying residual dsDNA in decellularised vessels, rat thoracic aortas were perfused with detergents (sodium dodecyl sulfate/sodium deoxycholate) with or without DNase I. Native aortas were used as control. Three different methods for purifying DNA (tissue lysate, salting out, silica-based solid-phase) were applied to assess dsDNA by fluorescence-based methods (PicoGreen, real-time QPCR, gel electrophoresis) or UV-spectrophotometry. Using tissue sections, H&E and fluorescent DAPI stainings for quantifying DNA were done additionally. RESULTS: DNase I-perfused aortas contained significantly lower amounts of dsDNA compared to native controls or detergent-perfused aortas. PicoGreen on tissue lysate showed 85.2% and 80.8% reduction in residual DNA; salted out DNA showed 90.3% and 84.6% reduction. Similarly, DAPI showed 91.1% and 82.4% reduction in DNA. QPCR reflected decreased concentration and fragmentation of residual DNA for salted out DNA, but not for solid-phase DNA. Gel electrophoresis using both purifications confirmed decreased DNA concentration and increased fragmentation, whereas spectrophotometry showed limited overall usability. CONCLUSION: Fluorescence-based DNA quantification using PicoGreen is useful for all three DNA purifications from decellularised aortas, and the only method applicable to tissue lysate. In turn, salted out DNA can be used reliably for PicoGreen, real-time QPCR and gel electrophoresis. Spectrophotometry is not recommended irrespective of the DNA purification. Complementary DAPI-based DNA quantification using tissue sections is advisable.