Intact marrow extraction enables nondecalcified bone marrow processing with improved molecular performance

The bone marrow is a complex microenvironment essential for hematopoietic and mesenchymal cell production, with a central role in physiological homeostasis and disease. Reliable preservation is therefore critical for accurate morphological and molecular analyses in both clinical and preclinical settings. Conventional decalcification methods maintain tissue structure but can compromise protein and nucleic acid integrity. Here, we present an intact marrow extraction (IME) method for recovering bone marrow cores from murine femora, enabling processing as soft tissue without decalcification while preserving native architecture. Morphology, protein localization, and ribonucleic acid (RNA) quality were compared between ethylenediaminetetraacetic acid (EDTA)-decalcified samples (n = 9) and nondecalcified IME samples (n = 9). Histological assessment showed comparable preservation of cellular morphology across methods. However, EDTA decalcification reduced the detection of adipogenic (LEPR) and osteogenic (SP7) markers by immunohistochemistry or immunofluorescence and was associated with diminished RNA signal by in situ hybridization, particularly for Ubc. While the RNA integrity (DV200) was similar, RNA yields were substantially higher in IME samples. Overall, IME preserves morphology and antigenicity while improving RNA yield and reducing processing time, providing a robust alternative to decalcification for integrated histological and molecular analyses of bone marrow and other calcified tissues.