Maintaining mRNA Integrity during Decalcification of Mineralized Tissues |
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| Authors: | Daniele Belluoccio Lynn Rowley Christopher B. Little John F. Bateman |
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| Affiliation: | 1. Murdoch Childrens Research Institute, Parkville, Victoria, Australia.; 2. Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia.; 3. Raymond Purves Bone and Joint Research Laboratories, Kolling Institute of Medical Research, St. Leonards, New South Wales, Australia.; University of Western Ontario, Canada, |
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| Abstract: | Biomineralization of the extracellular matrix occurs inappropriately in numerous pathological conditions such as cancer and vascular disease, but during normal mammalian development calcification is restricted to the formation of the skeleton and dentition. The comprehensive study of gene expression in mineralized skeletal tissues has been compromized by the traditional decalcification/fixation methods that result in significant mRNA degradation. In this study we developed a novel RNAlater/EDTA decalcification method that protects the integrity of the mRNA in mature mouse tibial epiphyses. Furthermore, this method preserves the tissue structure to allow histological sectioning and microdissection to determine region-specific gene expression, in addition to immuno- and in situ histology. This method will be widely applicable to the molecular analysis of calcified tissues in various pathological conditions, and will be of particular importance in dissection of the gene expression in mouse bone and joint tissues during development and in important clinical conditions such as arthritis. |
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