Recording and analysing DNA from osteocytes in resin-embedded bone samples

We report on a process to record the presence and the location of osteocyte nuclei using two nucleic staining dyes, Diamond™ Nucleic Acids Dye (DD) and DAPI (4',6-diamidino-2-phenylindole). Knowledge of the presence and number of osteocytes is key to any success in subsequent DNA profiling. Osteocytes are most numerous cells and thus the main source of DNA in bone samples, which can be preserved for histological analyses. Archived samples are either fixed in formalin or preserved in ethanol prior to embedding in resin. These resin-embedded samples are potentially used as ante mortem reference samples. Cases of a missing person investigation are one example where this type of preserved reference material may be of value. When resin is required for sample preservation it represents a problem for subsequent DNA profiling, if needed as a reference sample in human identification. It is essential therefore to remove the resin prior to DNA analyses as resin is a known inhibitor of DNA profiling. Current methods of resin removal are lengthy and require toxic chemicals. This report describes a simplified process to remove resin and visualise the location of nucleated osteocytes. Eight sections of bone samples at 5-µm thickness were stained with DD and DAPI. A further three samples were processed using a formalin-fixed method and three additional samples treated following an ethanol-preserved method (11 samples for both the formalin-fixed and 11 for the ethanol-preserved with eight in common). The location and number of nuclei could be recorded clearly due to the fluorescence created by the dye binding to DNA. The number of stained nuclei correlated with the mass of DNA isolated from the sections (r = 0.873, p = 1.21 × 10^-10). A significant difference between the degradation indices of two groups (p = 8.505 × 10^-5) showed that ethanol preservation is a preferred method to yield DNA of the quality needed for subsequent short tandem repeats (STR) profiling. Ten of the 11 samples isolated using the ethanol-preserved process recorded a complete STR profile (30/30 alleles), whereas eight of the formalin-fixed samples generated full profiles, and only one of the 11 samples amplified less than 23 alleles. Both the ethanol-preserved and formalin-fixed methods are an improvement on current methods by removing the need for strong solutes in resin removal, and the method leads to STR profiles from resin-embedded bone samples within 24 h.