Overcoming inconsistent DNA extraction recovery across tissue types in qPCR assays supporting biodistribution studies

AIMS: DNA biodistribution studies are essential in the development of cell and gene therapies. A key step of these studies is the extraction of vector DNA (vDNA) and genomic DNA (gDNA) from complex biological samples. In this work, key DNA extraction parameters were evaluated and optimized to ensure consistent and efficient DNA recovery. METHODS: Recovery was evaluated in multiple tissue types by spiking known amounts of vDNA and/or mouse gDNA into surrogate rabbit matrices, performing extraction using a DNA extraction kit under various conditions, and then quantifying the recovered DNA by qPCR and comparing with those spiked post extraction. RESULTS: Parameters including proteinase K incubation, RNase amount, DNA binding beads amount, and tissue input were systematically evaluated and found to play a significant role in recovery. By optimizing these parameters, consistently high recovery was achieved across various tissue types including brain, liver, and spinal cord from mouse, rabbit, and non-human primates. CONCLUSION: Proteinase K incubation, RNase amount, tissue input, and DNA binding beads volume were identified as key parameters influencing DNA recovery from tissue samples. The findings provide valuable insights for researchers to evaluate and optimize their own extraction protocols, ultimately enhancing the reliability and accuracy in supporting biodistribution studies.