Open AccessImprovements in cytogenetic slide preparation: Controlled chromosome spreading, chemical aging and gradual denaturing
Author(s) -
Henegariu Octavian,
Heerema Nyla A.,
Lowe Wright Lisa,
BrayWard Patricia,
Ward David C.,
Vance Gail H.
Publication year - 2001
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/1097-0320(20010201)43:2<101::aid-cyto1024>3.0.co;2-8
Subject(s) - chromosome , metaphase , fluorescence in situ hybridization , comparative genomic hybridization , fish <actinopterygii> , biology , in situ hybridization , karyotype , cytogenetics , molecular cytogenetics , multiplex , in situ , computational biology , genetics , chemistry , gene , gene expression , organic chemistry , fishery
Background Metaphase spreading is an essential technique for clinical and molecular cytogenetics. Results of classical banding techniques as well as complex fluorescent in situ hybridization (FISH) applications, such as comparative genomic hybridization (CGH) or multiplex FISH (M‐FISH), are greatly influenced by the quality of chromosome spreading and pretreatment of the slide prior to hybridization. Materials and Methods Using hot steam and a metal plate with a temperature gradient across its surface, a reproducible protocol for slide preparation, aging, and hybridization was developed. Results This protocol yields good chromosome spreads from even the most difficult cell suspensions and is unaffected by the environmental conditions. Chromosome spreads were suitable for both banding and FISH techniques common to the cytogenetic laboratory. Chemical aging is a rapid slide pretreatment procedure for FISH applications, which allows freshly prepared cytogenetic slides to be used for in situ hybridization within 30 min, thus increasing analytical throughput and reducing benchwork. Furthermore, the gradually denaturing process described allows the use of fresh biologic material with optimal FISH results while protecting chromosomal integrity during denaturing. Conclusion The slide preparation and slide pretreatment protocols can be performed in any laboratory, do not require specialized equipment, and provide robust results. Cytometry 43:101–109, 2001. © 2001 Wiley‐Liss, Inc.