A method for isolating high quality RNA from mouse cortical and cancellous bone

• Cortical and cancellous bone samples were isolated from mouse tibiae with negligible marrow contamination.
• Centrifugation and flushing to remove marrow were compared with histology and qPCR.
• High quality RNA can be isolated from cortical and cancellous bones separately, allowing gene expression comparison.
• Centrifugation removes marrow more efficiently than flushing and enhances detection of bone specific genes in cortical and cancellous bones.

The high incidence of fragility fractures in cortico-cancellous bone locations, plus the fact that individual skeletal sites exhibit different responsiveness to load and disease, emphasizes the need to document separately gene expression in cortical and cancellous bone. A further confounding factor is marrow contamination since its high cellularity may effect gene expression measurements. We isolated RNA from cortical and cancellous bone of intact mouse tibiae, and also after marrow removal by flushing or centrifugation. RNA isolated from cancellous bone by each method was sufficient for gene expression analysis. Centrifugation removed contaminating cells more efficiently than flushing, as indexed by histology and decreased expression of Icam4, a highly expressed erythroid gene. In contrast, centrifuged cortical bone had 12- and 13- fold higher expression of the bone-related genes Col1a1 and Bglap, while levels in marrow-free cancellous bone were 30- and 31-fold higher when compared to bone where marrow was left intact. Furthermore, cortical bone had higher expression of Col1a1 and Bglap than cancellous bone. Thus, RNA isolated by this novel approach can reveal site-specific changes in gene expression in cortical and cancellous bone sites.