A bone fragment-based protocol for RNA analysis of osteocyte-associated transcripts in surgically obtained human bone specimens.

Osteocytes play a central role in bone remodeling, mineral metabolism, and skeletal homeostasis; however, direct molecular analysis of human osteocytes remains technically challenging, as they are embedded within the mineralized bone matrix. Surgically obtained human bone specimens provide valuable material for studying human bone biology; however, surface-associated cells, marrow-derived cells, and adherent soft tissues can confound downstream transcript analysis. Here, we describe a bone fragment-based protocol for preparing surgically obtained human bone specimens for molecular analysis of osteocyte-associated transcripts. •The protocol consists of mechanical trimming, mincing into small bone fragments, repeated washing, and five sequential rounds of collagenase digestion to reduce non-osteocytic cellular components associated with bone surfaces and marrow spaces. The remaining mineralized bone fragments are then frozen in liquid nitrogen, cryogenically pulverized, and lysed in TRIzol™ reagent for total RNA extraction. •Histological validation using residual maxillary bone specimens showed that sequential collagenase digestion markedly reduced adherent soft tissue and extra-matrix nuclei while preserving osteocyte lacunar occupancy. Real-time PCR further showed marked enrichment of the osteocyte-associated transcripts SOST and DMP1 in residual bone fragments compared with those in Fraction 3-5. •This protocol provides a practical workflow for bone fragment-based RNA analysis focused on osteocyte-associated transcripts in human bone specimens.