Comparison of the Effectiveness of Four Commercial DNA Extraction Kits on Fresh and Frozen Human Milk Samples

For-profit donor human milk organizations have DNA-based proprietary methodology for testing incoming milk for adulteration with other species' milk. However, there is currently no standardized methodology for extracting DNA from human milk. Microbiome research has shown that DNA purity and quantity can vary depending on the extraction methodology and storage conditions. This study assessed the purity and quantity of DNA extracted from four commercially available DNA extraction kits-including one kit that was developed for human milk. This study was for method validation only. One donor provided a 90 mL human milk sample. The sample was aliquoted into 70 x 1 mL microcentrifuge tubes. Aliquots were randomized into one of three categories: fresh extraction, extraction after freezing, and extraction after purification and storage at room temperature. DNA was analyzed for purity and quantity using a NanoDrop Spectrophotometer. Results confirmed differences in DNA purity and quantity between extraction kits. The Plasma/Serum Circulating DNA Purification Mini Kit (Norgen Biotek, ON, Canada) provided significantly more DNA, and consistent purity as measured by 260/280 and 260/230 ratios. DNA quantity and purity were similar between fresh and frozen human milk samples. These results suggest that DNA purity and quantity is highest and most consistent when extracted from human milk using the Plasma/Serum Circulating DNA Purification Mini Kit amongst the kits tested in this study. Standardized methodology for extracting DNA from human milk is necessary for improvement of research in the field of human milk. To do this, future studies are recommended for optimization of DNA extraction from human milk using larger sample sizes and multiple donor parents.