title: Effect of bone decalcification procedures on DNA in situ hybridization and comparative genomic hybridization. EDTA is highly preferable to a routinely used acid decalcifier.
abstract: Decalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on the use of these techniques on archival decalcified bony material. In this study we compared the effects of two commonly used decalcifiers, i.e. , one proprietary, acid-based agent (RDO) and one chelating agent (EDTA), in relation to subsequent DNA ISH and CGH to bony tissues (two normal vertebrae, six prostate tumor bone metastases with one sample decalcified by both EDTA and RDO). We found that RDO-decalcified tissue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal status of EDTA-decalcified material of both control and tumor material. Gel electrophoresis revealed that no DNA could be successfully retrieved from RDO-treated material. Moreover, in contrast to RDO-decalcified tumor material, we detected several chromosomal imbalances in the EDTA-decalcified tumor tissue by CGH analysis. Furthermore, it was possible to determine the DNA ploidy status of EDTA- but not of RDO-decalcified material by DNA flow cytometry. Decalcification of bony samples by EDTA is highly recommended for application in DNA ISH and CGH techniques.
authors.affiliation: Department of Pathology, Erasmus University Rotterdam, Rotterdam, The Netherlands. Alers@PATH.FGG.EUR.NL
authors.forename: J C
authors.initials: JC
authors.lastname: Alers
authors.forename: P J
authors.initials: PJ
authors.lastname: Krijtenburg
authors.forename: K J
authors.initials: KJ
authors.lastname: Vissers
authors.forename: H
authors.initials: H
authors.lastname: van Dekken
mesh_terms: D001859:Bone Neoplasms / Q000737:chemistry / Q000556:secondary; D001842:Bone and Bones / Q000737:chemistry* / Q000187:drug effects; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D003648:Decalcification Technique; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D006851:Hydrochloric Acid / Q000494:pharmacology; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods*; D013131:Spine / Q000737:chemistry
chemical_list: D004247:DNA; D004492:Edetic Acid; D006851:Hydrochloric Acid