Application of droplet digital PCR to detect human Alu DNA elements in biodistribution assays in pre-clinical studies using rodents

Résumé

Abstract Biodistribution and engraftment assays for administered human cells in animal organs must be performed to ensure the safety and efficacy of the human cells in preclinical studies. Quantitative real-time polymerase chain reaction (qRT-PCR) is currently the standard method used to detect Alu DNA elements. The recently developed droplet digital PCR (ddPCR) method has been adopted in many biological fields, in which the small quantities of DNA must be detected. In this study, we evaluated whether ddPCR could replace qRT-PCR in biodistribution research. We applied ddPCR to detect human Alu DNA elements using various concentrations of genomic DNA (gDNA) extracted from human mesenchymal stem cells spiked with 100 ng of rat gDNA. We found that ddPCR could specifically detect human DNA and exhibited linearity within a concentration range of 10 ag–100 pg/20 µl. ddPCR was far more sensitive than qRT-PCR in measuring Alu elements, with a reliable lower limit of detection of 100 ag DNA/20 µl reaction. Additionally, the copy numbers of Alu elements were quantified using ddPCR in an ex vivo study examining DNA samples extracted from mouse spleen and lymph nodes that had been spiked with human natural killer cells; no human DNA was detected using qRT-PCR in this assay. Thus, we established a ddPCR method for detecting human gDNA in bio-distribution assays of human cells in rodents in preclinical studies.

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