Effect of bone decalcification procedures on DNA in situ hybridization andcomparative genomic hybridization: EDTA is highly preferable to a routinely used acid decalcifier

Decalcification is routinely performed for histological studies of bone-con taining tissue. Although DNA in situ hybridization (ISH) and comparative ge nomic hybridization (CGH) have been successfully employed on archival mater ial, little has been reported on the use of these techniques on archival de calcified bony material. In this study we compared the effects of two commo nly used decalcifiers, i.e., one proprietary, acid-based agent (RDO) and on e 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 ti ssue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal statu s of EDTA-decalcified material of both control and tumor material. Gel elec trophoresis revealed that no DNA could be successfully retrieved from RDO-t reated material. Moreover, in contrast to RDO-decalcified tumor material, w e detected several chromosomal imbalances in the EDTA-decalcified tumor tis sue by CGH analysis. Furthermore, it was possible to determine the DNA ploi dy status of EDTA- but not of RDO-decalcified material by DNA flow cytometr y. Decalcification of bony samples by EDTA is highly recommended for applic ation in DNA ISH and CGH techniques.