A method for isolating RNA from canine bone.

Extracting sufficient quantity and quality RNA from bone is essential for downstream application, such as transcriptomic sequencing, to evaluate gene expression. Isolation of RNA from bone presents a unique challenge owing to the hypocellular, brittle and mineralized matrix, which makes homogenizing the tissue difficult and provides little RNA to work with. Removal of contaminating tissue, such as bone marrow and connective tissue, is essential for isolating RNA that is unique to osteoblasts, osteoclasts and osteocytes. This study established a method to effectively isolate RNA from normal canine bone cells using the phalanges, without contamination from other tissue types, for downstream transcriptomic analysis. METHOD SUMMARY A combination of physical manipulation to remove exterior tissue, washing and centrifugation to remove cells and fat within the diaphysis, homogenization using a mortar and pestle on dry ice prior to bead dissociation, followed by acid guanidinium thiocyanate-phenol-chloroform extraction and column purification yielded sufficient quantity and quality RNA.