Laser capture microdissection-guided fluorescence in situ hybridization and flow cytometric cell cycle analysis of purified nuclei from paraffin sections.
Laser capture microdissection (LCM) has recently been identified as a quick, simple, and effective method by which microdissection of complex tissue specimens for molecular analysis can be routinely performed. Assessment of gene copy number by fluorescence in situ hybridization (FISH) is useful for the analysis of molecular genetic alterations in cancer. Unfortunately, the application of FISH to paraffin sections of tumor specimens is fraught with technical difficulty and potential artifacts. Our results demonstrate that LCM-microdissected nuclei are suitable for FISH gene copy analysis. Amplification of genes in cancer specimens can be detected as easily in LCM-prepared nuclei as in fresh nuclei from cancer tissue specimens. Furthermore, contamination of tumor specimens by normal cells can make interpretation of flow cytometric cell cycle analysis difficult. Our results show that LCM-microdissected nuclei can also be used for flow cytometric cell cycle and ploidy analysis. LCM/FISH offers the advantages of multicolor FISH in a morphologically defined cell population, without the technical problems of FISH performed on paraffin sections. This technique should further simplify the methodology required to perform copy number analysis of tumor suppressor or protooncogenes in archived cancer specimens. The use of LCM specimens will also improve the specificity and simplify the interpretation of flow cytometric cell cycle and ploidy analysis of breast cancer specimens.
DiFrancesco
L M
LM
Department of Pathology, University of Calgary, Alberta, Canada.
Murthy
S K
SK
Luider
J
J
Demetrick
D J
DJ
D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D002453:Cell Cycle; D002467:Cell Nucleus / Q000648:ultrastructure*; D005434:Flow Cytometry / Q000379:methods; D018628:Gene Dosage; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D016612:Paraffin Embedding; D011003:Ploidies