Fluorescence in situ hybridization assessment of c-myc gene amplification in breast tumor tissues.
This chapter details methods used for analysis of DNA copy-number changes in breast tumor tissues through the use of fluorescence in situ hybridization. The specific DNA probe described herein is the oncogene c-myc, although the tissue fluorescence in situ hybridization methodology presented is suitable for dual-color studies of most unique sequence and chromosome specific control probes. The breast tumor tissue sections are first deparaffinized in a solvent and clearing agent, and then pretreated with a protease to allow the target DNA within the breast tissue cells to be uncovered. This allows the DNA to be available for hybridization with the labeled c-myc probe. The tissue sections are then analyzed to assure that appropriate digestion of cellular material has been attained. The tissues are then denatured. The c-myc probe and control probe for the centromere of chromosome 8 are commercially available as differentially labeled and are cohybridized to the tissue and sealed beneath a cover slip in a humid chamber. They are incubated for 12-16 h. The cover slip is then removed, and the section is postwashed in 2X saline sodium citrate at 72 degrees C for 5 min and allowed to cool to room temperature in a detergent solution. The slides are then counterstained with 4',6-diamidino-2-phenylindole, and a cover slip is applied. The slides are then viewed with fluorescence microscopy using filters that allow the c-myc and chromosome 8 signals to be visualized. If possible, 50 cells are counted, and the data are expressed as number of c-myc signals/number of chromosome 8 signals.
Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC, USA.
Jan K
JK
Blancato
Mary Steele
MS
Williams
Robert B
RB
Dickson
D001943:Breast Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D005784:Gene Amplification; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000378:metabolism
D004273:DNA, Neoplasm; D016271:Proto-Oncogene Proteins c-myc