Hair root sonication washing impact on nuclei counts.

Abstract

In a previous study, hair samples were washed by sonication in Terg-a-zyme™ (Alconox, White Plaines, NY, USA) as part of the DNA extraction protocol. The sonication wash step was deemed necessary in the previous study because the DNA extracts could be used for both mitochondrial (mt) and nuclear(nu) DNA analyses. In the current study, the impact of the sonication wash on the persistence of nuclei was assessed. Scalp hair roots were stained using the DNA binding dye 4', 6-diamidino-2-phenylindole (DAPI) and visible nuclei were counted. Hair roots that contained <50 visible nuclei were primarily used to monitor the change in the number of visible nuclei due to sonication. Hair roots with a larger number of nuclei counts (e.g., >100) were also included as comparative data. The selected hair samples were washed by sonication in Terg-a-zyme™. The number of nuclei detected in unwashed DAPI-stained hair roots was compared with the number in washed DAPI-stained hair roots. Intricacies that impeded visible nuclei counting in the same hair root were observed, such as orientation of the mounted hair root, hair root morphology, turgescence, and folding of soft tissue. Despite these challenges, this study showed the sonication wash of hair roots containing <50 visible nuclei might lead to a reduction of nuclei available for DNA extraction and short tandem repeat (STR) typing. However, for laboratories performing only nuDNA analysis, the sonication could be replaced with a less aggressive hair washing method such as briefly vortexing at low speed in saline followed by an ethanol rinse.